GIFT  OF 

Pacific  Coast 


fllOLOCY  UBRftRY 


CLINICAL 
LABORATORY 
FOR 


BY 

ANNA  L.  GIBSON,  R.N. 

Matron  Superintendent  of  the  Collis  P.  Huntington 

Memorial  Hospital^  Harvard  Medical  School^ 

Boston,  Mass. 


REVISED    EDITION 


WHITCOMB  &  BARROWS 

BOSTON,    1922 


£5 


COPYRIGHT  1916  AND  1922 

BY 
ANNA    L.    GIBSON 


THOMAS  TODD  Co.,  PRINTERS 
14  Beacon  Street,  Boston,  Massachusetts 

MADE    IN   U      S.    A. 


TO 

EMMA    M.  NICHOLS,  R.N. 

Formerly  Superintendent  of  Nurses •,  The  Boston  City  Hospital,  Boston,  Mass 

IN    APPRECIATION    OF   THE 
INSPIRATION,  KNOWLEDGE,  AND   TRAINING 

WHICH   I    RECEIVED 
FROM   ASSOCIATION    WITH   HER 


743540 


PREFACE 

THIS  book  owes  its  existence  to  the  frequent  request 
on  the  part  of  graduate  nurses,  whom  it  has  been  my 
pleasure  to  instruct  in  laboratory  technic  during  the  past 
three  years,  for  a  simple,  comprehensive  text-book,  that 
the  nurses  might  be  able  to  grasp  the  principles  of  clinical 
laboratory  technic. 

The  book  was  originally  compiled  as  a  handbook  for 
practical  clinical  laboratory  work,  since  no  single  text- 
book covered  the  work. 

The  arrangement  of  the  several  chapters  has  worked 
itself  out  from  a  series  of  lessons  which  give  simple  and 
reliable  methods.  By  these  methods  information  may  be 
obtained  without  unnecessary  detail  which  requires  a 
considerable  knowledge  of  general  chemistry  and  elab- 
orate apparatus. 

Standard  works  on  Bacteriology,  Chemistry,  Hema- 
tology,  Histology,  and  Parasitology  have  been  consulted 
freely,  and  references  are  given  at  the  end  of  each  chapter 
from  which  more  detailed  information  may  be  obtained. 

A  great  deal  of  the  material  and  the  drawings  have 
been  taken  from  my  notebook. 

Grateful  acknowledgment  is  made  to  Dr.  Thomas 
Ordway,  Dean  of  Albany  Medical  College,  with  whom 
I  have  had  the  privilege  of  working  the  past  three 
years  at  the  Huntington  Hospital ;  to  Dr.  Ellis  Kellert, 
Director  of  the  Bender  Laboratory,  Albany,  New  York, 
who  gave  me  valuable  assistance  the  two  years  we 
were  associated  at  the  Huntington  Hospital ;  and  to 
Dr.  Ernest  Tyzzer,  Assistant  Professor  of  Pathology, 


vi  Preface 

Harvard  Medical  School,  and  Director  of  Huntington 
Hospital,  Harvard  Medical  School. 

ANNA  L.  GIBSON,  R.  N. 
BOSTON,  MASSACHUSETTS. 

September,  1915. 

NEW  PREFACE 

WITH  the  aim  of  keeping  this  book  abreast  of  present- 
day  laboratory  technic,  the  entire  work  has  been  carefully 
revised  and  considerable  new  matter  has  been  added. 
Hospital  schools  of  nursing  are  being  called  upon  to  pro- 
duce specialists  in  many  fields  of  endeavor,  and  there 
should  be  a  broader  and  more  systematic  study  of  the 
various  branches  relating  to  nursing. 

There  is  no  more  important  subject  than  clinical  labo- 
ratory technic,  nor  one  more  far-reaching  in  its  influence 
on  the  ultimate  success  or  failure  of  the  work  of  a  nurse. 

This  book  was  not  written  with  the  thought  of  mak- 
ing nurses  diagnosticians,  but  to  help  them  to  have  a  more 
intelligent  and  more  thorough  understanding  of  this  sub- 
ject in  its  relation  to  nursing. 

I  have  had  the  advantage,  as  formerly,  of  the  help  and 
advice  of  professors  in  Harvard  Medical  School.  I  am 
especially  indebted  to  Dr.  Robert  B.  Greenough,  Assistant 
Professor  of  Surgery  at  Harvard  Medical  School  and 
Director  of  the  Cancer  Commission  of  Harvard  Univer- 
sity, for  encouragement  in  my  teaching  of  this  subject; 
to  Dr.  Francis  Peabody  and  Dr.  George  Minot,  Profes- 
sors of  Medicine,  Harvard  Medical  School,  for  instruc- 
tion in  laboratory  technic ;  also  to  various  authors  whose 
works  I  have  consulted,  and  to  publishers  who  have  so 
willingly  granted  me  permission  to  use  illustrations  from 
their  books.  ANNA  L.  GIBSON,  R.  N. 

1922. 


CONTENTS 

CHAPTER  PAGE 

I.     LABORATORY  EQUIPMENT         .        .        .        .        .        .  i 

II.     THE  MICROSCOPE      .        .        .        .        .        .  16 

III     URINE        . •     . .        .  -      .'-     . 20 

IV.     FECES         .       '.        . 56 

V.     GASTRIC  CONTENTS 70 

VI.     SPUTUM      .        .      ..        .        .        i       v       .        .        .84 

VII.    THE  BLOOD       .        .        .        .        .        .        „    •    .        .      90 

VIII.     BACTERIA  . 141 

IX.     CULTURE  MEDIA       . 166 

X.    BODY  FLUIDS    .        . 180 

XI.     MILK 187 

XII.     PREPARATION  OF  TISSUE         •     •  .        .        .        ,        .  192 

APPENDIX 197 


vii 


CHAPTER  I 
LABORATORY 


Glassware  {   ^  *\*,,arlJ  >0°' 

It  is  very  important  that  all  glassware  used  in  the 
laboratory  should  be  made  of  Bohemian  or  Jena  glass. 
Many  test  tubes  are  made  of  the  ordinary  glass,  which  is 
silicate  of  calcium  and  sodium,  and  sometimes  lead  oxide 
is  used  instead  of  calcium  carbonate. 

This  glass  is  easily  acted  upon  by  chemical  substances, 
and  should  not  be  subjected  to  heat. 

Tubes,  flasks,  and  beakers  should  be  made  of  potassium 
carbonate  glass  —  Bohemian  glass  —  as  this  glass  is  char- 
acterized by  its  great  hardness,  difficult  fusibility,  and  by 
its  resistance  to  the  action  of  chemical  substances,  sudden 
changes  of  temperature,  and  high  steam  pressure. 

The  best  glass  tubing  and  rods  are  made  of  Jena 
glass,  as  it  is  chemically  superior  to  the  Bohemian  glass, 
more  resistant  to  acidulous  fluids  and  sudden  changes  of 
temperature. 

Care  of  Glassware 

All  glassware  used  in  the  laboratory  work  must  be 
thoroughly  clean  before  using.  New  glassware  should 
be  placed  in  0.5%  solution  of  nitric  acid  to  remove  the 
alkali  frequently  present;  thoroughly  rinse  in  running 
water. 

Glass  slides  are  cleaned  by  immersing  in  cleaning  solu- 
tion, then  washing  in  water;  dry  with  a  towel  and  flare 


2  t   Clinical  Laboratory  Technic 

both  sides  over  a  Bunsen  flame.    Oil  which  has  dried  on 
slides  can  be  removed  with  xylol. 

Old  test  tubes  containing  culture  media  should  be  re- 
sterilized  for  one  hour,  or  boiled  for  one  hour  in  a  5  % 
solution  of  soda;  this destroys  the  bacteria  and  loosens 
the  material  is?  -the  tubes. 

Test  tubes  'anil'  flasks  are  dried  in  the  autoclave,  then 
plugged  with  non-absorbent  cotton  and  sterilized  one-half 
hour  at  15  pounds  pressure. 

A  good  cleaning  fluid  is  made  as  follows : 

Potassium  bichromate  60  c.c. 

Concentrated  sulphuric  acid  300  c.c. 

Water  400  c.c. 

Dissolve  the  potassium  bichromate  in  water  with  heat. 

Cool,  then  add  slowly  the  sulphuric  acid. 

Glass  Droppers  and  Capillary  Pipettes 

Take  a  piece  of  tubing  and  heat  it  in  the  middle  of  a 
Bunsen  flame,  revolving  the  tubing  while  heating;  and 
when  it  becomes  soft  in  the  center,  remove  from  the  flame 
and  with  a  steady  pull  separate  the  ends.  Cool,  file,  and 
break  off.  Flare  the  rough  ends  in  the  flame. 

Glass  Stirring  Rods 

Take  a  piece  of  glass  rod,  file  off  the  desired  length, 
then  round  off  the  rough  ends  in  the  flame  by  constant 
rotation. 

Weights  and  Measures 

The  Analytical  Balance.  The  poise  in  the  ordinary 
balance  is  not  disturbed  by  slight  variations  of  weight, 
but  in  chemical  analysis  a  more  sensitive  instrument  is 


Laboratory  Equipment 


necessary.  The  beam  is  made  as  light  as  possible,  the 
bearings  sharp  and  hard,  the  adjustments  capable  of  being 
brought  to  the  last  degree  of .  refinement  and  provided 
with  appliances  for  arresting  its  action  at  will.  It  is  in- 
closed in  a  glass  case  for  protection  against  dust,  moisture, 
and  currents  of  air. 

The  beam  is  divided  by  notches  into  tenths,  and  carries 
weights  shaped  as  rid- 
ers, and  these  riders 
lessen  in  value  as  they 
are  moved  towards  the 
center. 

A  rider  weighing 
.01  gram  in  the  pan 
weighs  .09  gram  at  first 
notch  from  the  pan, 
.08  gram  at  the  second. 

Large  brass  weights 
equal     grams;     large 
platinum  weights,  0.5   gram ;  small  weights,   .05   gram ; 
and  the  rider,  .01  gram. 

The  gram  is  the  unit  of  weight  and  equals  the  weight 
of  i  c.c.  of  distilled  water  at  4°  C. 

I  kilogram  =  1,000  grams  =  100,000  centigrams  = 
1,000,000  milligrams. 

I  kilogram  =  2.20462  pounds  =  35.2739  ounces  — 
15,432.35  grains. 

Always  lift  the  weights  with  the  forceps  provided. 

A  watch  glass  is  used  as  a  receptacle  for  reagents 
weighed.  First  ascertain  the  weight  of  the  glass  and  add 
the  amount  to  the  required  amount  of  the  reagent. 

Note.  Objects  to  be  weighed  should  be  placed  on  the  left-hand 
pan,  and  the  weights  on  the  right-hand  pan. 


ANALYTICAL  BALANCE 


4 


Clinical  Laboratory  Technic 


A  meter  equals  39.37  inches. 

A  cubic  meter  is  the  unit  of  space  for  the  number  of 
organisms  in  air.  It  contains  1,000  liters.  It  is  equal 
to  1.308  cubic  yards  or  35.316  cubic  feet.  1,000  cubic  feet, 
the  unit  of  space  in  disinfection,  is  equal  to  28.3  plus  cubic 
meters. 

A  cubic  centimeter  is  the  unit  of  space  for  organisms 


a 
sq.m.m. 


c.c 


sq.inch. 


METRIC ''SYSTEM  2 

INCHES  n  21  31 

I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  1  I  I  I  I  I  I  I  I  I  I  I  I  I  I  1  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  J  J  I  I  1 

COMPARISON  OF  INCHES  AND  CENTIMETERS 

in  water,  milk,  vaccines,  etc.  There  are  approximately 
16  drops  in  i  c.c. 

Cubic  millimeter  is  the  unit  of  space  for  blood  cells. 
There  are  1,000  cubic  millimeters  in  i  cubic  centimeter, 
and  1,000,000  cubic  millimeters  in  i  liter. 

A  liter  is  the  unit  of  space  for  volumetric  solutions. 
It  contains  1,000  cubic  centimeters,  and  is  equal  to  1.0567 
quarts  or  33.8  ounces.  A  liter  of  distilled  water  equals 
I  kilogram. 


Laboratory  Equipment 


Decimal  Table 

Length 

1,000 

kilometer 

100 

hectometer 

10 

decameter 

I 

Meter 

O.I 

decimeter 

0.0  1 

centimeter 

O.OOI 

millimeter 

The  arc  is  the  unit  of  surface  and  is  the  square  of  ten 
meters. 


Weight  Capacity 

kilogram  kiloliter 

hectogram  hectoliter 

decagram  decaliter 

Gram  Liter 

decigram  deciliter 

centigram  centiliter 

milligram  milliliter  or  cubic  centimeter 


Reagents 

The  reagent  bottles  should  be  made  of  Jena  glass,  which 
is  free  from  lead  and  other  impurities,  and  these  bottles 
should  be  fitted  with  ground  glass  stoppers.  All  reagents 
should  be  chemically  pure  (C.  P.)- 

Liquid  Reagents 

Nitric  acid,  C.  P.  (HNO3)  ;  acetic  acid,  (HC2H3O2)  ; 
sulphuric  acid,  C.  P.  (H2SO4)  ;  hydrochloric  acid,  C.  P. 
(HC1)  ;  ammonic  hydrate,  (NH4OH)  ;  sodic  hydrate, 
(NaOH),U.  S.  P. 

Solid  Reagents 

Cupric  sulphate,  caustic  soda,  sodium  chloride,  potas- 
sium iodide,  potassium  chromate,  ammonium  sulphate, 
magnesium  sulphate,  ammonium  chloride,  sodium  acetate, 
potassium  ferrocyanide,  potassium  acetate,  guaiac,  ben- 
zidin,  potassium  chlorate,  picric  acid,  citric  acid,  lead 
acetate,  sulphanilic  acid,  sodium  nitrite,  sodium  carbon- 
ate, mercuric  chloride,  potassium  bromide,  sodium  nitro- 
prusside,  alphol  naphthol,  phenylhydrazin  hydrochlorate, 


6  Clinical  Laboratory  Technic 

di-methyl-amino-azo-benzine,  di-methyl-paraphenylene-di- 
amine,  Eosin  Gruber,  w.  g. 

The  different  alcohols  used  in  making  up  the  various 
reagents  are:  absolute  alcohol,  which  contains  not  less 
than  99%  by  weight  of  pure  ethyl  alcohol,  C2H5OH ; 
alcohol  of  a  stated  percentage,  e.  g.,  50%,  means  a  mix- 
ture with  water  which  contains  the  stated  percentage, 
e-  £•>  5°%>  by  volume  of  pure  ethyl  alcohol;  methyl 
alcohol,  a  pure  substance,  CH3OH,  prepared  by  the  puri- 
fication of  commercial  wood  spirit ;  and  methylated  alcohol 
or  methylated  spirit,  which  may  be  used  instead  of  pure 
ethyl  alcohol  in  preparing  solutions  of  various  percent- 
ages of  alcohol.  Methylated  alcohol  is  a  mixture  of 
19  parts  of  ethyl  alcohol  and  i  part  commercial  methyl 
alcohol.  Commercial  methyl  alcohol  is  impure  and  must 
not  be  used  in  making  Eosin-Azur,  Louis  Jenner's,  Leish- 
man's,  Wright's,  or  Romanowsky  stains. 

The  amounts  of  distilled  water  and  absolute  alcohol 
required  to  produce  saturated  solutions  of  dyes  in  common 
use  are  indicated  in  the  following  table : 

Alcohol  (ex.) 

7 

2-5 

7 

I 
I 

7 
10 

To  Remove  Glass  Stoppers 

If  there  is  any  difficulty  in  removing  the  glass  stoppers 
from  the  reagent  bottles,  they  are  easily  loosened  by 
gently  tapping  the  neck  of  the  bottle  with  another  piece 
of  glass. 


Dye 

Water 

Bismarck  Brown 

I 

7 

Fuchsin  (Basic) 

I 

10 

Gentian  Violet 

I 

7 

Hematoxylin 

I 

2 

Methyl  Violet 

I 

5 

Methylene  Blue 

I 

7 

Thionin  Blue 

I 

5 

Laboratory  Equipment 
Table  of  Equivalents 


Liquids 

Approximate 

Accurate 

i  minim 

=         0.06 

c.c. 

0.06  1 

c.c. 

16      " 

==         i. 

c.c. 

i  fl.  dram 
i  fl.  ounce 

=         4- 
=       30. 

c.c. 
c.c. 

3.697 
29-574 

c.c. 
c.c. 

i  pint 
i  gallon 

=     500. 
==  4,000. 

c.c. 
c.c. 

473-197 
3,785. 

c.c. 
c.c. 

Solids 

i  kilogram 

=            2.2 

Ibs.  av. 

I  gram 

=     is- 

grains 

15432 

grains 

I  milligram 

=    A 

grain 

0.015. 

\  grain 

Solutions 

Approximately  correct  solutions  are  made  by  using  the 
following  method : 

For  i -i, ocx)  (TO%)  use  TS  grains  to  a  liter. 
For  i-ioo  (i%)  use  5  grains  to  the  ounce. 
EXAMPLES,    i.  Make  2,500  c.c.  of  a  1-500  solution  of 
potassium  permanganate.    15  grains,  or  i  c.c.,  to  500  c.c. 
is  a  1-500  solution.    If  15  grains,  or  i  c.c.,  is  used  to  every 
500  c.c.,  for  2,500  c.c.  we  would  use  as  many  c.c.  as  there 
are  500  in  2,500,  or  5  c.c.  (75  grains). 

2.  Make  500  c.c.  of  i -10,000  solution  caustic  potash. 
If  it  takes  15  grains  to  1,000  c.c.  to  make  a  1-1,000  solu- 
tion, to  make  a  1-10,000  solution,  which  is  10  times  weaker, 
take  -^Q  of   15  grains,  or  1.5  grains,  for  1,000  c.c.  of  a 
1-10,000  solution,  and  for  500  c.c.  of  a  i-io,ooc  solution 
take  ^  of  15  grains,  or  0.75  grain. 

3.  Make  75  c.c.  of  a  \%  solution  of  acetic  acid.     \% 
equals  1-300.     75  c.c.  -f-  300  c.c.  =  .25  c.c.     If  i  c.c.  is 
used  to  every  300  c.c.,  to  75  c.c.  we  would  use  \  as  much ; 
therefore  with  a  pipette  measure  0.25  c.c. 


8  Clinical  Laboratory  Technic 

Saturated  Solution 

The  solubility  of  many  metallic  salts,  acids,  alkalies, 
sugars,  and  organic  products  is  very  great,  yet  there  is 
a  limit  beyond  which  it  is  not  possible  to  dissolve  a  solid 
in  liquid.  This  limit  is  the  point  of  saturation. 

Concentrated  Solution 

When  we  speak  of  a  concentrated  solution  we  mean 
the  amount  of  the  solute  in  a  given  quantity  of  the 
solvent. 

Standard  Solution 

A  standard  solution  is  one  whose  concentration  is 
known.  This  can  be  made  of  any  strength.  To  obtain 
a  standard  solution  use  a  solution  of  sodium  hydroxide 
of  known  strength.  This  gives  a  standard  of  hydrogen 
fixing  power.  Units  of  this  solution  will  be  equivalent 
to  definite  amounts  of  acid  in  the  solution  neutralized. 

;* 

Normal  Solution 

A  normal  solution  is  a  standard  solution  which  con- 
tains in  one  liter  the  hydrogen  equivalent  in  grams  of 
the  active  reagent.  A  normal  volumetric  solution  is 
made  by  dissolving  the  hydrogen  equivalent  or  atomic 
weight  divided  by  its  valence  in  distilled  water  and 
making  its  volume  up  to  one  liter.  The  molecular  weight 
in  grams  of  a  base,  salt  or  acid,  is  divided  by  the  valence, 
and  the  valence  of  a  base  is  the  number  of  hydroxyls 
combined  with  it ;  the  valence  of  an  acid  is  the  number 
of  replaceable  hydrogen  atoms  which  it  contains. 

If  the  molecule  of  the  element  is  univalent,  one  liter 
will  contain  the  weight  in  grams  equal  to  the  molecular 
weight  of  the  element;  if  bivalent,  a  weight  in  grams 


Laboratory  Equipment  9 

equal  to  one-half  its  molecular  weight ;  if  trivalent,  a 
weight  equal  to  one-third  its  molecular  weight. 

Common  Elements 


(1920)       Symbols 

Atomic 

Symbols 

Atomic 

Weights 

Weights 

Aluminum 

Al 

27.1 

Gold 

Au 

197.2 

Antimony 

Sb 

120.2 

Hydrogen 

H 

1.008 

Arsenic 

As 

74.96 

Iodine 

I 

126.92 

Barium 

Ba 

137.37 

Iron 

Fe 

55.84 

Bismuth 

Bi 

208. 

Lead 

Pb 

207.20 

Boron 

B 

10.09 

Magnesium 

Mg 

24.32 

Bromine 

Br 

79.92 

Mercury 

Hg 

200.6 

Cadmium 

Cd 

II2.4 

Nitrogen 

N 

14.008 

Calcium 

Ca 

40.07 

Oxygen 

0 

16. 

Carbon 

C 

12.005 

Phosphorus 

P 

31.04 

Chlorine 

Cl 

3546 

Potassium 

K 

39.10 

Chromium 

Cr 

S2- 

Silver 

Ag 

107.88 

Cobalt 

Co 

58.97 

Sodium 

Na 

23.00 

Copper 

Cu 

63.57 

Sulphur 

S 

32.06 

Fluorine 

F 

10. 

EXAMPLE.  NaOH  is  univalent.  Na  =  23,  O  =  16, 
H  =  1.008.  The  sum  of  these  atomic  weights  equals 
40.008.  Dissolve  40.008  grams  of  NaOH  in  distilled 
water  and  make  up  to  one  liter. 

H2SO4  is  bivalent.  II2  =  2.016,  S  =  31.83,  O4  = 
63.52.  The  sum  of  these  atomic  weights  equals  97.366. 
As  it  has  two  replaceable  H  atoms,  it  would  contain  in 
one  liter  of  water  one-half  this  number,  or  48.683  grams 
of  absolute  H2SO4. 

Solutions  of  this  strength  are  designated  by  the  capital 
letter  N ;  of  twice  this  strength,  by  2N ;  one-half  or  one- 
tenth,  by  N/2  or  N/io.  Equal  volumes  of  normal  solu- 
tions react  together  completely.  One  liter  of  normal 
NaOH  or  KOH  will  neutralize  one  liter  of  normal 
H2SO4  or  HNO3. 


io  Clinical  Laboratory  Technic 

METHOD.  Clean  volumetric  flasks  and  beakers  with 
cleaning  solution;  rinse  thoroughly  in  running  water, 
twice  with  distilled  water,  and  then  with  a  small  portion 
of  the  solution  they  are  to  contain. 

Pick  out  pure  crystals  of  oxalic  acid,1  place  in  a  watch 
glass,  and  weigh  with  analytical  balances,  the  weight  of 
the  glass  having  been  previously  ascertained. 

The  molecular  weight  of  oxalic  acid  is  126.048.  As  it 
is  diabasic,  divide  by  two.  63.024  grams  are  necessary  to 
make  one  liter  of  N  solution.  To  make  N/io,  take  6.3024 
grams. 

Pour  the  crystals  into  a  dry  beaker,  rinsing  off  the 
watch  glass  with  distilled  water ;  stir  with  a  glass  rod 
until  dissolved. 

Place  the  glass  rod  in  the  neck  of  a  liter  volumetric 
flask  and  carefully  pour  the  oxalic  solution  from  the 
beaker.  Rinse  the  beaker  several  times  with  distilled 
water,  then  wash  off  the  rod  and  fill  the  flask  from  a  wash 
bottle  of  distilled  water  up  to  the  mark;  the  last  few 
drops  can  be  blown  from  the  wash  bottle. 

As  the  inside  neck  of  the  flask  is  wet  from  the  addition 
of  water,  a  filter  paper  which  has  been  rolled  up  is  in- 
serted and  rotated  until  the  neck  is  dry,  being  careful  not 
to  touch  the  fluid. 

Stopper  and  thoroughly  mix. 

Having  N/io  oxalic  acid,  N/io  sodium  hydroxide  is 
prepared  as  follows:  Sodium  hydroxide  is  very  hygro- 
scopic, therefore  a  normal  solution  cannot  be  accurately 
prepared  by  weight.  Weigh  out  5  grams  and  dissolve  in 
1,100  c.c.  of  water.  Titrate  with  the  oxalic  solution. 

If  accurately  measured  should  give  N/io  solution  in  which  the 
error  is  less  than  i  %. 


Laboratory  Equipment 


n 


B 


A.  Graduated  burette  and  support. 

B.  Graduated  pipette. 

C.  Volumetric  flask. 

D.  Erlenmeyer  flask. 


Indicators  are  substances  which  assume  a  deep  color 
in  the  presence  of  certain  other  substances,  or  change 
sharply  from  one  deep  color  to  another. 

EXAMPLES.  Phenolphthalein,  a  very  feeble  acid,  is 
colorless  in  the  presence  of  acids  (i.e.,  hydrion),  and  red 
in  the  presence  of  alkalies  (i.e.,  hydroxidion).  Litmus 
is  red  with  acids,  and  blue  with  alkalies.  The  change  of 
color  depends  upon  a  chemical  interaction. 


12  Clinical  Laboratory  Technic 

Titration 

A  definite  amount  of  oxalic  acid  solution  is  measured 
with  a  graduated  pipette  and  placed  in  a  flask.  Add 
50  c.c.  of  distilled  water,  then  3  drops  of  alizarin  or 
phenolphthalein,  as  an  indicator  that  the  point  of  neutral- 
ization or  end  of  the  reaction  can  be  accurately  deter- 
mined. Phenolphthalein  is  used  when  neither  ammonia 
nor  bicarbonates  are  titrated.  In  titrating  acid  and  alkali, 
always  run  the  alkali  into  the  acid. 

EXAMPLE.  Pipette  10  c.c.  of  the  oxalic  acid  solution 
into  an  Erlenmeyer  flask;  do  not  blow  out  the  amount 
left  in  the  end  of  the  pipette.  Pour  50  c.c.  of  distilled 
water  into  the  flask  and  add  3  drops  of  phenolphthalein. 
Rinse  out  a  burette  with  distilled  water  and  with  the 
sodium  hydroxide  solution,  then  fill  with  the  solution 
until  it  rises  above  the  zero  mark.  A  few  c.c.  are  run  out 
until  the  mark  is  reached.  Place  the  flask  of  oxalic  solu- 
tion on  a  filter  paper  for  a  white  background  and  run  in 
the  sodium  hydroxide  solution,  a  few  c.c.  at  a  time,  shak- 
ing the  flask  gently.  Add  the  sodium  solution  until  there 
is  a  distinct  pinkish  color,  which  is  the  end  of  the  reaction. 

If  9.8  c.c.  of  the  sodium  hydroxide  solution  were  re- 
quired to  produce  a  distinct  pink  color,  it  is  stronger  than 
the  N/io  oxalic  acid  solution,  as  only  10  c.c.  would  have 
been  necessary  if  the  sodium  solution  had  been  N/io. 
Therefore  9.8  c.c.  of  the  sodium  solution  are  equivalent 
to  10  c.c.  of  the  N/io  oxalic  solution.  The  sodium  hy- 
droxide must  be  diluted  in  the  proportion  of  9.8  to  10. 
Measure  exactly  1,000  c.c.  of  the  too  concentrated  sodium 
hydroxide  solution  and  add  20  c.c.  of  distilled  water. 


Laboratory  Equipment 


Equivalent  Fahrenheit  and  Centigrade  Tables 


AUTOCLAVE  TEMPERATURE 

Sterile  dressings,  media,  disin- 
fection of  spore-bearing  bac- 
terial contamination 


C. 

125 
1 20 

H5 
no 

105 

100 


F. 
258 
250 
240 
230 

220 

212 


FREEZING  TEMPERATURE 

Preserving  biological  products ; 
post-mortem  material 

C.  F. 


40 


4 
3 

2 

I 
0 


BODY  TEMPERATURE 

Growth  of  important  patho- 
genic organisms 

C.  F. 


40 

39 
38 
37 


104 


98.2 


ROOM  TEMPERATURE 

Culturing  gelatin 

(melting  point,  25°  C.) 

as  in  water  work 


C. 

14 
23 

22 
21 
20 


F. 

75 


69.8 
68 


PARAFFIN  AND  PASTEURIZING 

Bacterial  vaccines  and 
paraffin  bath 

C.  F. 

80  176 

75 

70  158 

65 

60  140 

Fahrenheit  scale  is  divided  into  equal  divisions  or 
degrees,  the  lowest  of  which  is  a  mixture  of  equal  parts 
of  sal-ammoniac  and  snow,  and  the  highest,  the  boiling 


14  Clinical  Laboratory  Te clinic 

point  of  pure  water;  the  freezing  point  of  water  on  this 

scale  is  32°. 

Centigrade  scale  is  divided  into  100  equal  parts,  or 
degrees,  the  space  of  expansion  from  the 
freezing  point  to  the  boiling  point  of  pure 


joo_: 


aio- 

water.     The   number   of   degrees   between 
the  boiling  point  and  freezing  point  in  Centi- 
grade is  100,  and  in  Fahrenheit  it  is  212  —  32 
or  180.    EXAMPLE.   100:1  80::  degree  to  be 
g     converted  :X.    By  division  with  20,   5:9:: 
£     degree  to  be  converted  :X;  i.e.,  the  degree 
g     to  be  converted  is  multiplied  with  9,  the  re- 
g     suit  divided  with  5,  and  32  added  to  the 
result. 


H 

u 


p  Centigrade  X  0 

-^—^  --  \-  32  =  Fahrenheit. 

<  .    c  ' 


I          Fahrenheit  —  32  X  5        ^     ,.       -, 
g  _^ ^  ==  Centigrade. 

es  Q 


Laboratory  Rules 

1.  All  possible  cleanliness  should  be  ob- 
j_         served  in  the  care  of  apparatus. 

2.  The  hands  should  be  washed  with  a 
disinfectant  after  working  with  pathogenic 
bacteria,  and  then  with  soap  and  water. 

3.  Pencils  and  labels  should  never  be  moistened  with 
the  lips. 

4.  Discarded  cultures  should  be  covered  with  a  disin- 
fectant, then  resterilized. 

5.  Culture  media  should  be  put  in  receptacles  provided 
for  that  purpose,  and  not  in  the  sink. 


Laboratory  Equipment  15 

6.  Pipettes  which  have  been  used  to  handle  infectious 
material  should  be  placed  in  a  glass  receptacle  containing 
cleaning  fluid. 

7.  Any  infectious  material  dropped  on  the  table  or  floor 
should  be  immediately  wiped  with  a  disinfectant. 

8.  All  bottles  should  be  plainly  labeled. 

9.  All  cultures  and  tissue  specimens  should  be  labeled 
with  the  patient's  name,  source,  and  date. 

10.  Sterilize  the  platinum  loop  before  and  after  use. 

11.  When  using  the  autoclave,  see  that  there  is  suffi- 
cient water  before  turning  on  the  pressure. 

12.  Whenever   material   is   placed   in   the   centrifuge 
tubes,  see  that  they  balance  evenly. 


CHAPTER  II 


THE  MICROSCOPE 

One  of  the  most  important  pieces  of  apparatus  used  in 
the  laboratory  is  the  microscope. 

i.  The  microscope  consists  of  a  tube  160  millimeters 
(6.4  in.)  long,  having  two  systems 
of  lenses,  which  conduct  the  rays  as 
they  pass  from  the  objective  to  the 
oculars. 

2.  The  Ocular,  or  eyepiece,  is  at 
the  upper  end  of  the  tube.    There  are 
various  oculars  and  they  are  num- 
bered from  one  to  ten,  the  magnify- 
ing power  of  the  ocular  increasing 
as  the  number  advances. 

3.  The  Objective  is  a  system  of 
converging  lenses  at  the  lower  end 
of  the  tube,  which  forms  a  magnified 
inverted  image  of  the  object. 

4.  The  Stage,  with  clips  to  hold  a 
slide  in  position  during  examination. 

5.  The  Reflector  or  small  mirror  has  two  sides,  a  con- 
cave and  a  plane  mirror.     The  reflector  directs  the  rays 
of  light  upward  through  the  object  in  the  optical  axis  of 
the  microscope. 

6.  The   Sub-Stage  Condenser  is  a  system  of  lenses 
between  the  stage  and  the  mirror.    These  lenses  collect 
and  condense  the  rays  coming  from  the  reflector  so  that 

16 


MICROSCOPE 


The  Microscope  17 

they  are  focused  upon  the  object,  thus  giving  a  brilliant 
illumination. 

7.  The  Iris  Diaphragm  controls  the  intensity  of  the 
illumination  and  is  just  below  the  sub-stage  condenser. 
The  gradations  of  light  are  obtained  by  means  of  a  small 
lever. 

8.  The  Coarse  Adjustment  is  a  rack  and  pinion  mech- 
anism which  rapidly  raises  and  lowers  the  barrel  and  its 
attachments. 

9.  The  Fine  Adjustment  is  just  below  the  coarse  ad- 
justment.    This   very   gradually   raises   and  lowers  the 
barrel  in  order  to  obtain  exact  focus. 

Illumination 

Direct  sunlight  should  be  avoided.  North  light  is  to  be 
preferred,  as  it  is  the  most  uniform  and  steady.  The 
character  and  color  of  artificial  light  is  much  improved 
by  inserting  a  piece  of  blue  glass  between  the  concave 
reflector  and  the  object. 

Focusing 

Focus  the  body  tube  down  until  the  objective  touches 
the  cover  glass,  then  with  the  eye  to  the  eyepiece  focus 
up  carefully. 

Often  one  acquires  the  habit  of  using  only  the  right 
or  left  eye  for  microscopic  work,  but  it  is  better  to  learn 
to  use  both.  Both  eyes  should  be  open  when  using  the 
microscope. 

Always  examine  a  specimen  first  with  a  low  power  and 
then  with  a  high  power  objective. 

When  the  oil-immersion  objective  is  used,  a  drop  of 
cedar  oil  is  placed  between  the  slide  and  the  end  of  the 


1 8  Clinical  Laboratory  Technic 

objective,  and  the  objective  is  brought  into  contact  with 
this  and  the  observation  made  through  the  oil.  The  oil- 
immersion  objective  is  so  constructed  that  when  in  use 
the  pencil  of  light  passing  through  the  object  to  the  objec- 
tive traverses  only  media  of  the  same  refractive  index, 
and  cedar  oil  has  the  same  refractive  index  as  glass. 

The  oil  acts  as  a  third  lens  and  increases  the  magnify- 
ing power  of  the  microscope. 

A  fine  hair  cemented  into  one  of  the  oculars  serves  as 
a  pointer  and  is  of  great  aid  in  singling  out  some  special 
object  of  interest. 

Objects  are  often  accidentally  present  in  microscopical 
preparations,  such  as  air  bubbles.  If  small,  they  may  look 
entirely  dark ;  if  large,  they  are  clear  in  the  middle,  with 
a  broad,  dark  border,  due  to  refraction  of  the  light. 

Linen  fibers,  well-defined,  rounded,  slightly  twisted. 

Cotton  fibers,  longer,  broader,  but  thinner  and  more 
twisted  than  linen. 

Woolen  fibers  and  hairs  have  the  same  structure, 
although  the  wool  is  finer  and  is  curled. 

Dust  of  the  room,  showing  groups  of  black  particles  of 
carbon  (soot),  and  shed  epithelium-cells  derived  from 
the  epidermis. 

Molds,  with  long,  branching  filaments  (hyphae),  and 
the  torula-like  particles  (spores)  from  which  hyphae  may 
in  some  instances  be  seen  sprouting. 

Yeast  particles  or  torulse:  each  torula  contains  a  clear 
vacuole  and  has  a  well-defined  outline,  due  to  a  membrane. 

Starch  granules :  fine  concentric  lines  are  seen  in  the 
granules,  arranged  around  a  minute  spot  which  is  placed 
near  the  smaller  end  of  the  granule. 

Lens  paper  or  fine  linen  moistened  with  xylol  should 


The  Microscope  19 

be  used  to  wipe  the  objectives,  and  this  must  be  applied 
carefully,  as  the  lenses  are  mounted  in  balsam. 

The  microscope  should  be  lifted  by  some  one  of  its  solid 
parts,  and  never  by  any  part  above  the  stage. 

The  glass  surfaces  should  never  be  touched  with  the 
ringers,  and  the  metal  parts  should  be  kept  free  from 
liquids,  especially  acids  and  alkalies. 


CHAPTER  III 
URINE 

The  volume  of  urine  excreted  by  normal  individuals 
varies  between  1500  and  2000  c.c. 

The  condition  of  the  kidneys  and  their  capability  for 
work  are  determined  by  accurate  chemic  and  microscopic 
examination,  and  such  information  is  of  great  importance 
in  judging  the  diagnosis  and  prognosis  of  disease. 

Anuria  is  the  suppression  of  the  secretion  in  the  kidneys 
or  obstruction  in  the  urinary  tract. 

Polyuria  is  the  increase  in  the  elimination  of  urine  as 
a  whole,  both  fluid  and  solid. 

Hydruria  is  the  increase  in  the  total  twenty-four  hour 
amount  out  of  proportion  to  the  solids. 

Oliguria  is  the  diminution  in  the  total  excretion  of  the 
urine. 

Dysuria  is  painful  or  difficult  urination. 

Consistence 

Clear  and  liquid,  frequently  turbid  and  viscid  (ropy). 
In  normal  urine  this  turbidity  is  caused  by  precipitated 
phosphates  or  urates ;  in  abnormal  urine  it  results  from 
casts,  cells,  etc.  The  slight  cloud-nubecula  which  urine 
develops  on  standing  is  formed  by  bacteria,  desquamated 
cells,  and  traces  of  mucus. 

Odor 

Aromatic,  due  to  volatile  acids.  The  odor  of  ammonia 
is  important  only  in  fresh  urine.  If  free  ammonia  is 

20 


Urine  21 

present,  moistened  red  litmus  paper  will  turn  blue  when 
held  over  the  mouth  of  a  tube  in  which  perfectly  fresh 
urine  is  boiled.  Acetone  odor  indicates  diabetes  mellitus ; 
putrid  odor  denotes  pus. 

Collection  for  Analysis,  Twenty-four  Hour  Amount 

Different  voidings  may  vary  greatly  in  chemical  com- 
position; therefore  quantitative  tests  are  of  no  value 
unless  a  sample  of  the  mixed  24  hour  amount  be  used. 

At  8  A.M.  the  bladder  is  emptied  and  the  urine  thrown 
away;  then  save  all  urine  voided  until  8  A.M.  the  next 
day,  voiding  it  exactly  at  8  and  adding  to  the  amount 
saved.  Collect  this  24  hour  amount  in  a  perfectly  clean, 
two-liter  bottle,  and  keep  well  corked  in  a  cool  place.1 

Urine  begins  to  decompose  a  few  hours  after  it  is 
voided;  therefore  add  5  c.c.  of  toluol,  one  of  the  most 
satisfactory  preservatives,  which  apparently  interferes 
with  none  of  the  tests.  Dust,  feces,  and  expectoration 
interfere  with  analysis,  and  care  should  be  taken  that 
these  do  not  enter  the  bottle. 

A  single  specimen  may  be  obtained  for  qualitative 
examination,  and  as  there  is  a  marked  variation  in  the 
urine  at  different  times  a  day,  a  specimen  should  be  taken 
several  hours  after  a  meal,  when  it  contains  the  greatest 
amount  of  abnormal  elements. 
Color 

Normal  urine  varies  from  straw  to  amber  color,  and 
this  color  is  derived  from  urochrome  and  other  pigments. 

1  Chloral  and  chloroform  reduce  Fehling's  solution  and  should  not 
be  used  if  the  urine  is  to  be  tested  for  glucose. 

Formaldehyde  coagulates  albumin  and  reduces  Fehling's. 

Thymol  is  used  for  saccharine  urine,  as  it  has  no  reducing 
action  (t  gr.  to  I  fl.  oz.  of  urine).  Boric  acid  may  be  used,  5  grs. 
to  4  fl.  oz.  of  urine. 


22 


Clinical  Laboratory  Technic 


Halliburton's  Table 


Color 

Nearly  color- 
less. 


Dark  yellow  to 

brown-red. 

i 

Milky. 


Orange. 

Red  or 
reddish. 


Brown  to 
brown-black. 


Greenish  yel- 
low, brown 
approaching 
black. 

Dirty  green  or 
blue. 


Brown-yellow 
to  red-brown, 
becoming 
blood-red 
upon  adding 
alkalies. 


Cause  of  Color 
Dilution  or  diminution 
of  normal  pigment. 


Increase  of  normal  or 
occurrence  of  patho- 
logic pigment. 

Fat  globules. 
Pus  corpuscles. 

Excreted  drugs,  e.g., 

Unchanged   hemoglobin. 

Pigment  in  food   (log- 
wood,   madder,    bilber- 
ries, fuchsin). 

Hematin. 
Methemoglobin. 
Melanin. 

Hydrochinone  and  cate- 
chol. 

Bile  pigment. 


A  dark  blue  scum  on 
surface,  with  a  blue  de- 
posit, due  to  an  excess 
of  indigo-forming  sub- 
stances. 

Substances   contained  in 
senna,   rhubarb,   and 
chelidonium,  which  are 
introduced  into  the 
system. 


Pathologic  Conditions 

Various  nervous  con- 
ditions, hydruria, 
diabetes  insipidus, 
granular  kidney. 

Acute  febrile  diseases. 


Chyluria. 

Purulent  disease  in 
urinary  tract. 

Santonin,  chryso- 
phanic  acid. 

Hemorrhage  or 
hemoglobinuria. 


Small  hemorrhages. 
Methemoglobinuria. 
Melanotic  sarcoma. 
Carbolic  acid  poison- 
ing. 

Jaundice. 


Cholera,  typhus;  seen 
especially  when  the 
urine  is  putrefying. 


Urine  23 

• 

Urine  may  darken  on  exposure  to  the  air,  owing  to  the 
presence  of  alkapton,  a  decomposition  product  of  the  pro- 
teins found  as  a  crystalline  body  in  the  urine. 

Reaction 

In  the  majority  of  cases  the  mixed  twenty- four  hour 
amount  is  acid  to  litmus.  The  reaction  may  undergo 
marked  changes  in  both  physiological  and  pathological 
conditions. 

Animal  food  produces  acid  urine,  and  a  vegetable  diet 
may  give  a  neutral  or  alkaline  urine. 

The  reaction  varies  according  to  the  time  of  day  the 
urine  is  voided.  It  may  be  neutral  for  some  time  after  a 
meal,  or  alkaline  to  litmus,  as  at  that  time  the  secretion 
of  hydrochloric  acid  into  the  stomach  during  the  process 
of  digestion  reduces  the  store  of  acids  in  the  body.  This 
change  is  known  as  the  "alkaline"  tide. 

Urine  may  become  alkaline  owing  to  the  conversion  of 
urea  into  ammonium  carbonate  by  micro-organisms. 

Microscopically  such  a  urine  shows  amorphous  phos- 
phates, ammonium  magnesium  crystals,  and  ammonium 
urate  crystals. 

If  a  urine  is  alkaline  immediately  after  voiding,  it  may 
be  due  to  a  fixed  alkali  or  ammonia,  and  ammoniacal 
fermentation  is  ordinarily  due  to  cystitis. 

Acid  fermentation  occurs  in  urine  having  a  normal 
acidity  when  voided,  which  upon  standing  becomes 
strongly  acid.  The  sediment  of  such  urine  shows  acid 
urates,  uric  acid,  calcium  oxalate  crystals,  and  fungi. 

Blue  litmus  paper  is  changed  red  by  acid  urine.  Red 
litmus  paper  is  changed  blue  by  alkaline  urine. 

When  urine  changes  blue  litmus  paper  red  and  red 


24  Clinical  Laboratory  Technic 

9 

litmus  paper  blue,  it  has  an  amphoteric  reaction,  due  to 
the  simultaneous  presence  in  the  urine  of  acid  and  alkali 
salts. 

Specific  Gravity 

The  specific  gravity  of  a  body  is  its  weight,  as  compared 
with  the  weight  of  an  equal  bulk  of  a  standard  body  taken 
as  a  unity. 

The  specific  gravity  of  normal  urine  varies  between 
1,015  and  1,025.  This  means  that,  taking  distilled  water 
at  77°  F.  as  i,  each  cubic  centimeter  of  urine  weighs 
1,015  or  1,025  grams.  The  urine  should  be  at  the  same 
temperature.  If  it  is  not,  to  every  7°  above  normal  add 
one  unit  of  the  last  order  to  the  last  reading;  to  every 
7°  below  normal  subtract  one  unit  of  the  last  order. 

EXAMPLE.  The  urinometer  is  graduated  for  77°  F. 
and  reads  1,018  at  63°  F. ;  then  the  specific  gravity  at 
77°  F.  would  be  1,018  —  0.002,  or  1,016.  77°  —  63°  = 
14°,  and  one  unit  is  subtracted  to  every  7°  below  normal; 
14°  divided  by  7°  =  2°,  therefore  2°  X  o.ooi  (one  unit 
of  the  last  order)  =  0.002.  1,018  —  0.002  =  1,016. 

The  relative  proportion  of  solid  matter  in  the  urine  is 
shown  by  the  specific  gravity,  and  by  knowing  the  total 
amount  in  twenty- four  hours  an  approximate  idea  of  the 
absolute  solids  is  obtained  by  multiplying  the  last  two 
figures  of  the  specific  gravity  by  Long's  coefficient,  *.  e., 
2.6.  The  solid  content  in  1,000  c.c.  is  obtained  by  multi- 
plying the  last  two  figures  of  the  specific  gravity  observed 
at  25°  C.  by  2.6. 

EXAMPLE.  Sp.  gr.  =  1,020.  20  X  2.6  =  52  grams, 
the  amount  of  solids  in  1,000  c.c.  of  urine.  The  total 


Urine  25 

amount  in  twenty-four  hours  is  1,500  c.c. ;  therefore  it 
will  contain  1,000 : 1,500 :  :$2  :x,  or  78  grams, 

Meat,  drugs  (potassium  acetate),  albumin,  and  diabetes 
mellitus  raise  the  specific  gravity. 

There  is  a  decrease  in  chronic  interstitial  nephritis  and 
diabetes  insipidus. 

METHOD.  The  urinometer  glass  is  filled  three-fourths 
full  of  urine ;  then  introduce  the  urinometer  and  wait  until 
it  finds  the  correct  level  before  reading  the  scale.  The 
last  mark  seen  below  the  surface  (the  meniscus)  is  the 
correct  reading  of  the  scale,  and  this  should  be  read 
through  the  fluid  from  below  upward. 

When  the  amount  of  urine  is  so  small  .that  it  is  neces- 
sary to  dilute  to  get  sufficient  to  take  the  specific  gravity, 
add  four  times  as  much  water  as  urine,  making  five 
volumes. 

EXAMPLE.  To  one  volume  of  urine  add  four  volumes 
of  water,  making  five  volumes.  If  the  specific  gravity  of 
this  mixed  fluid  reads  1,005,  then  that  of  the  urine  will 
be  1,000  plus  (5  X  5),  or  1,025. 

Albumin 

Albuminuria  is  a  condition  in  which  serum  albumin  or 
serum  globulin  appears  in  the  urine,  and  these  two  protein 
substances  are  of  the  greatest  pathologic  import. 

In  the  accidental  albuminuria  the  albumin  is  not  ex- 
creted by  the  kidneys,  but  arises  from  the  blood,  lymph, 
or  some  albumin  containing  exudate  coming  in  contact 
with  the  urine  at  some  point  below  the  kidneys. 

The  renal  type  is  the  more  serious,  as  the  albumin  is 
secreted  by  the  kidneys. 


26  Clinical  Laboratory  Technic 

Nitric  Acid  Test.  (Heller's) 

Put  5  c.c.  of  filtered  urine  in  a  test  tube  and  introduce 
5  c.c.  of  concentrated  nitric  acid  very  carefully  with  a 
pipette  to  the  bottom,  forming  an  underlayer. 

Various  colored  zones,  due  to  the  presence  of  indican, 
bile  pigment,  or  to  the  oxidation  of  other  organic  urinary 
elements,  may  form,  but  these  should  never  be  mistaken 
for  the  white  zone  which  alone  indicates  serum  albumin, 
serum  globulin,  albuminose,  and  peptone. 
Magnesium  Mixture  for  Turbid  Urine 

Filter  10  c.c.  of  urine;  add  sufficient  magnesium  mix- 
ture to  make  the  urine  alkaline.  After  testing  with  litmus 
paper,  add  a  few  drops  of  acetic  acid.    Filter,  and  test  for 
albumin  with  nitric  acid. 
Albumose  Test  for  "Bence- Jones"  Bodies 

To  10  c.c.  of  urine  add  5  drops  of  acetic  acid ;  boil,  then 
filter.  On  the  surface  of  5  c.c.  of  nitric  acid  in  a  test  tube 
gently  pour  some  of  the  cooled  filtrate.  A  white  ring  at 
the  junction  of  the  fluids  indicates  the  presence  of  albu- 
mose,  and  this  will  disappear  on  warming,  to  reappear 
again  on  cooling. 
Coagulation  or  Boiling  Test 

Fill  the  test  tube  half  full  of  filtered  urine  and  gently 
heat  the  upper  half  to  boiling,  being  careful  that  this  half 
of  the  fluid  does  not  mix  with  the  lower  half.  A  turbidity 
indicates  serum  albumin,  serum  globulin,  or  phosphates. 
Acidify  the  urine  with  5  drops  of  acetic  acid;  the  turbid- 
ity, if  due  to  phosphates,  will  disappear.  If  albumose  is 
present,  a  white  cloud  appears  on  heating,  disappears 
on  boiling,  reappears  on  cooling. 


Urine  27 

Quantitative  Test  for  Albumin.    (Esbach's  Method) 
This  test  is  made  by  means  of  a  standard  graduated 
glass  tube  or  albuminometer. 

Add  10  drops  of  a  10%  solution  of  FeCl3  to  the  urine 
before  introducing  the  Esbach's  reagent ;  warm  the  tube. 
Fill  the  tube  with  urine  to  the  letter  U,  then  add  Esbach's 
reagent  to  R;  close  the  tube  with  a  stopper  and  invert 
several  times.  Let  the  tube  stand  2  hours,  then  read  off 
the  number  of  grams  of  albumin  to  the  liter,  as  will  be 
indicated  by  the  number  on  the  side  of  the  tube  on  a 
level  where  the  albumin  settles.  If  the  urine  is  alkaline, 
add  a  few  drops  of  acetic  acid.  Urine  heavy  with 
albumin  should  be  diluted  with  i  or  2  volumes  of  water. 
EXAMPLE.  The  protein  precipitate  is  level  with  the 
figure  2  of  the  graduated  scale,  therefore  the  urine  con- 
tains 2  grams  of  the  protein  to  the  liter,  or  .2%  (the 
amount  of  protein  in  per  cent). 

Test  for  Lead 

Evaporate  2  liters  of  urine  to  a  tenth  of  its  volume. 
Add  an  equal  volume  of  20%  hydrochloric  acid,  and 
3  grams  of  potassium  chlorate.  Heat  the  mixture  on 
the  water  bath  to  60°  C.  Add  3  rrams  of  potassium 
chlorate  as  soon  as  the  evolution  of  chlorine  has  ceased. 
Repeat  this  until  the  fluid  no  longer  gives  off  the  fumes 
of  chlorine  on  the  further  addition  of  the  chlorate.  Water 
may  be  added  if  the  liquid  becomes  too  concentrated. 
Cool,  dilute  with  water,  then  filter.  Examine  the  filtrate 
for  lead  with  hydrogen  sulphide,  sulphuric  acid,  and 
potassium  bichromate.  If  lead  is  present  there  will  be 
precipitates  of  black  lead  sulphide,  white  lead  sulphate, 
and  yellow  lead  chromate. 


28  Clinical  Laboratory  Techmc 

Albumin.    (Goodman  and  Steam's  Method) 

To  5  c.c.  of  phosphotungstic  acid  solution  add  2  c.c.  of 
filtered  urine  with  a  pipette  graduated  in  tenths.  Shake 
after  addition  of  each  tenth;  add  urine  until  a  whitish 
cloud  appears.  The  number  of  tenths  is  read  off  and 
expressed  in  terms  of  100  c.c.  EXAMPLE.  If  it  takes  i  c.c. 
of  diluted  urine  (i-io)  there  is  o.oooi  gram  albumin, 
or  i  gram  in  100  c.c. ;  if  0.7  c.c.  of  diluted  urine,  then 
0.07  c.c.  of  undiluted  urine  equals  o.oooi  gram  albumin. 
7  c.c.  =  .01  gram  albumin,  700  c.c.  =  i  gram.  700:1.0 
::ioo:x,  or  .142%. 

Sugar 

Very  small  amounts  of  glucose  probably  occur  in  every 
normal  urine.  In  diabetes  mellitus  there  is  a  permanent 
or  persistent  glucose  excretion.  Glucosuria  is  a  transient 
type  and  accompanies  various  disorders,  particularly 
cerebral  digestive  affection,  certain  forms  of  poisoning, 
morphine,  carbon  monoxid,  chloral  hydrate,  oil  of  turpen- 
tine, corrosive  sublimate,  amyl  nitrite,  and  prolonged 
hunger.  Sometimes  perfectly  healthy  people  excrete 
glucose  in  urine  after  too  abundant  ingestion  of  sugar. 

Urine  containing  much  glucose  presents  a  light  or  pale 
color  and  has  a  high  specific  gravity. 

The  most  common  sugar  occurring  in  urine  is  glucose. 
Levulose  is  sometimes  present  with  glucose.  Lactose  is 
occasionally  found  in  the  urine  of  nursing  women ;  laiose, 
maltose,  and  pentose  are  rare.  If  albumin  is  present,  it 
should  be  removed  before  testing  for  sugar.  This  is 
done  by  boiling  and  adding  a  few  drops  of  acetic  acid, 
and  filtering. 


Urine  29 

Benedict's  Test  responds  to  all  the  carbohydrates, 
and  every  urine  should  be  tested  for  sugar  by  this  re- 
duction test. 

Fermentation  Test  responds  only  to  glucose,  levulose, 
and  maltose. 

Phenylhydrazin  Test  responds  to  glucose,  levulose, 
maltose,  lactose,  and  pentose.  This  is  a  very  delicate 
and  reliable  test. 

o  • 

Polarimetric  Test:  glucose,  lactose,  and  maltose,  and 
to  a  slight  extent  pentose,  rotate  the  ray  of  polarized  light 
to  the  right;  while  levulose,  /3-oxybutyric  acid,  and  gly- 
curonates  rotate  polarized  light  to  the  left.  Normal  urine 
is  often  slightly  dextro-rotary. 

Benedict's  Qualitative  Test  for  Glucose 

Heat  to  boiling  5  c.c.  of  Benedict's  Solution  in  a  test 
tube  (put  a  glass  bead  in  the  tube  to  prevent  bumping). 
Add  8  drops  of  urine  and  boil  2  minutes.  If  more  than 
.2  to  .3%  of  sugar  is  present,  the  solution  will  be  filled 
with  a  greenish,  yellow,  or  reddish  precipitate.  With 
smaller  amounts  of  sugar  the  precipitate  appears  only 
on  cooling. 

The  reagent  is  made  as  follows: 
86  grams  sodium  citrate 
50  grams  anhydrous  sodic  carbonate 
400  c.c.  distilled  water 

Stir  until  entirely  dissolved,  then  add  8.6  grams  copper 
sulphate  that  has  been  dissolved  in  50  c.c.  of  hot  water. 
Pour  the  copper  solution  slowly,  and  with  stirring,  into 
the  alkaline  citrate  solution.  Make  up  to  one  liter. 


Clinical  Laboratory  Technic 


Benedict's   Method  for  the   Quantitative   Estimation   of   Sugar.- 
From  GradwohFs  "  Blood  and  Urine  Chemistry" 


Urine  31 

Benedict's  Quantitative  Estimation  o£  Glucose 

.    Dilute  10  c.c.  of  urine  with  distilled  water  up  to  100  c.c. 
Mix,  then  transfer  to  a  burette. 

Pipette  25  c.c.  of  the  volumetric  solution  into  a  Jena 
flask  (150  c.c.  capacity).  Add  5  to  10  grams  of  sodium 
carbonate  and  i  gram  of  powdered  pumice. 

Heat  the  mixture  to  boiling.  Run  urine  in  rapidly 
from  the  burette  until  a  white  precipitate  begins  to  form, 
then  more  slowly  with  continuous  boiling,  until  the  blue 
color  entirely  disappears.  This  is  the  end  point. 

EXAMPLE.  If  8  c.c.  of  diluted  urine  were  used  in 
reducing  25  c.c.  of  Benedict's  Volumetric  Solution,1  then 
0.8  c.c.  of  undiluted  urine  was  required,  and  this  amount 
contained  0.050  gram  glucose.  The  percentage  in  the 
sample  would  be  calculated  thus : 

0.8 :  0.050: :  100:  x,  which  equals  6.25% 
If  1,400  c.c.  of  urine  were  voided  in  twenty-four  hours, 
then  0.8: 0.050: :  1,400:  x  or  87.50  grams  glucose. 

Benedict's  Volumetric  Solution 
18.0  grams  copper  sulphate. 
100   grams    anhydrous   or    double   the   quantity  of 

crystallized  sodium  carbonate. 
200  grams  of  sodium  or  potassium  citrate. 
125  grams  of  potassium  sulphocyanate. 
5  c.c.  of  a  $%  solution  of  potassium  ferrocyanide. 
Make  up  to  I  liter  with  distilled  water. 
Dissolve  the  copper  sulphate  separately  in  150  c.c.  of 
distilled  water  and  then  add  slowly  with  constant  stir- 
ring to  a  filtered  solution  (about  800  c.c.)  of  the  other 
ingredients.     Make  up  to  I  liter. 

iNote.  The  25  c.c.  of  copper  solution  are  reduced  by  exactly 
50  mg.  of  glucose. 


32  Clinical  Laboratory  Technic 

Fermentation  Test 

This  test  is  important  because  the  fermentable  sugars- 
are  the  pathological  ones.  Carbon  dioxide  and  alcohol 
are  formed  in  the  fermentation  of  sugar,  and  the  produc- 
tion of  alcohol  and  the  disappearance  of  sugar  lower 
the  specific  gravity. 

The  urine  should  be  sterilized  by  boiling,  and  if  not 
already  acid,  acidified  with  hydrochloric  or  tartaric  acid. 
Cool,  and  determine  the  specific  gravity.  Add  a  small 
piece  of  yeast  about  the  size  of  a  bean.  Place  200  c.c.  of 
this  urine  in  a  flask  and  stopper  sufficiently  close  to  pre- 
vent the  escape  of  alcohol.1  Let  the  flask  of  urine  stand 
in  a  warm  room  24  hours,  then  test  with  Fehling's  solu- 
tion. If  sugar  is  still  present,  let  it  stand  tor  another 
24  hours.  If  the  reducing  substance  is  still  present,  it  is 
not  a  fermentable  sugar.  Record  the  specific  gravity. 

It  has  been  found  that  a  decrease  of  o.ooi  (a  fall  of 
one  point)  in  the  specific  gravity  corresponds  to  0.23% 
of  sugar  in  the  urine  tested. 

EXAMPLE.  The  specific  gravity  of  the  urine  before 
fermentation  is  1,030.  After  the  fermentation  is  com- 
pleted, the  specific  gravity  is  1,008,  a  decrease  of  22 
points ;  and  this  multiplied  by  0.23  gives  the  amount, 
5.06%,  of  sugar  in  the  urine  tested. 

Phenylhydrazin  Test 

Sugars  form  ozazones  when  treated  with  phenyl- 
hydrazin. 

1  Great  care  should  be  taken  not  to  plug  the  fia.sk  so  tightly  as  to 
cause  an  explosion.  This  method  is  a  much  more  accurate  procedure 
if  the  saccharometer  of  Lohnstein  is  used. 


Urine 


33 


PHENYLHYDRAZIN  TEST  FOR  SUGARS 

In  a  test  tube  put  nearly  ^  in.  (i  gm.)  of  phenylhydrazin  hydro- 
chloric!, an  equal  quantity  of  powdered  sodium  acetate,  and 
enough  of  the  suspected  fluid  to  half-fill  the  tube.  The  acetate 
dissolves  as  the  tube  is  heated.  Boil  for  2  minutes  and  examine 
after  20  minutes,  or,  if  hurried,  examine  a  drop  under  the  micro- 
scope at  once  without  a  cover-glass.  In  2  or  3  minutes  the  crystals 
form:  a,  Sheaves  and  stars  of  needles — glucosazone;  b,  rosettes 
of  lance-shaped  crystals  —  maltosazone;  ct  spicules  in  burr-like 
clusters  —  lactosazone. 


34  Clinical  Laboratory  Technic 

Benzidin  Test  for  Occult  Blood 

Add  2  c.c.  of  a  saturated  solution  of  benzidin,  in  alco- 
hol or  acetic  acid,  to  2  c.c.  of  3%  hydrogen  peroxide  and 
i  c.c.  of  urine.  Blood  is  indicated  by  the  appearance  of 
a  green  or  blue  color. 

Gerhardt's  Test  for  Acetone 

To  10  c.c.  of  urine  in  a  test  tube  add  10  drops  of  acetic 
acid,  then  add  10  drops  of  freshly  prepared  sodium  nitro- 
prusside  solution1  and  mix;  carefully  overlay  with  2  c.c.  . 
of  concentrated  ammonia. 

If  acetone  be  present,  a  violent- red  ring  will  develop 
at  the  point  of  contact.  The  amount  of  acetone  will  be 
increased  in  fever,  with  starvation,  with  purely  meat  diet, 
in  diabetes  mellitus,  in  certain  forms  of  digestive  disturb- 
ance, and  in  some  cases  of  carcinoma. 

Acetone  is  a  physiological  as  well  as  a  pathological 
constituent  of  the  urine. 

Acetone  is  said  to  be  excreted  under  the  following 
pathological  conditions : 

Diabetes  mellitus 

Pneumonia 

Nephritis 

Deranged  digestive  function 

Fasting 

Autointoxication 

Anesthesia 

Scarlet  and  typhoid  fevers 

Phosphorous  poisoning 

Grave  anemias 

1  Sodium  nitropmsside  solution:  To  5  grams  sodium  nitroprusside 
add  5  c.c.  water;  or  a  crystal  of  sodium  nitroprusside  may  be  added  to 
the  acidified  urine. 


Urine  35 

Normal  adults  on  a  mixed  diet  excrete  3  to  15  mg.  of 
combined  acetone  and  acetoacetic  acid  per  day;  more  is 
considered  pathological. 

In  severe  diabetic  acidosis  6  grams  or  more  may  be 
excreted,  Acidosis  is  due  mainly  to  a  disturbance  in 
the  metabolism  of  fats. 

Diacetic  Acid  Test 

To  5  c.c.  of  urine  add  an  excess  of  a  10%  solution 
of  ferric  chloride.  A  Bordeaux-red  color  indicates  dia- 
cetic  acid.  If  this  color  is  masked  by  the  precipitate 
of  ferric  phosphate,  the  fluid  should  be  filtered.  The  red 
color,  if  due  to  diacetic  acid,  disappears  on  heating. 
The  presence  of  antipyrin,  aspirin,  phenacetine,  salicylic 
acid,  and  sodium  acetate  give  a  similar  red  color,  which 
does  not  disappear  en  heating. 

Test  for  Indican  or  Potassium  Indoxyl  Sulphate 

The  test  for  indican  is  based  upon  the  fact  that  an  excess 
of  HC1  will  liberate  the  indoxyl,  and  by  the  addition  of 
an  oxidizing  agent  this  is  converted  into  indigo  blue,  and 
finally  this  can  be  recognized  in  small  amounts  by  extrac- 
tion from  the  bulk  of  urine  with  chloroform.  The  pres- 
ence of  more  than  a  trace  indicates  the  existence  of  un- 
desirable decomposition  in  the  intestinal  tract. 

METHOD.  Add  10  c.c.  of  urine  to  the  same  amount  of 
Chemically  Pure  concentrated  HC1.  Mix  and  add  3  drops 
of  freshly  prepared  \%  solution  of  potassium  perman- 
ganate. If  indican  is  present,  a  purplish  cloud  will  form. 
Then  add  a  few  drops  of  chloroform  and  then  a  few  more 
drops  of  permanganate  solution.  Shake  vigorously  and 


36  Clinical  Laboratory  Technic 

the  color  will  change  to  a  deep  blue,  due  to  the  precipita- 
tion of  indican  by  the  chloroform. 

Test  for  Melanin 

In  cases  of  melanotic  sarcoma,  the  urine  treated  with 
iron  chlorid  assumes  a  deep  black  color. 

Urochromogen  and  Diazo  Tests  in  Tuberculosis 

(Journal  A.  M.  A.,  October  10,  1914) 
Place  i  c.c.  of  limpid  urine  diluted  with  3  c.c.  of  water 
in  two  test  tubes.  To  the  first  tube  add  3  drops  of  1-1,000 
solution  of  potassium  permanganate;  the  other  tube  use 
as  a  control.  A  distinct  canary  yellow  indicates  a  positive 
reaction. 

Iodine  Test  for  Bile 

To  10  c.c.  of  urine  in  a  test  tube  add  2  c.c.  of  tincture 
of  iodine  (dilute  i-io  in  alcohol).  The  presence  of  bili- 
rubin  is  indicated  by  a  distinct  emerald  green  ring  at  the 
point  of  contact. 

Urobilin  Test 

Fill  a  test  tube  with  12  c.c.  of  urine,  add  i  drop  of  con- 
centrated hydrochloric  acid,  shake  gently,  and  allow  to 
separate  by  standing.  Pour  off  the  supernatant  fluid  and 
add  three  times  its  volume  of  alcohol.  To  this  alcoholic 
extract  add  i  drop  of  a  $%  solution  of  zinc  chloride  and 
i  drop  of  ammonium  hydroxide.  Zinc  hydrate  will  be 
precipitated  and  should  be  filtered  off.  A  green  fluores- 
cence indicates  the  presence  of  urobilin. 

Urobilin  is  an  abnormal  product  and  is  increased  in 
carcinoma,  appendicitis,  Addison's  disease,  acute  infec- 
tious diseases,  and  pancreatic  disease. 


Urine  37 

Urophaein 

To  10  c.c.  of  urine  add  2  c.c.  of  H2SO4.  A  brownish 
red  color  indicates  the  presence  of  urophsein. 

Urochrome 

Add  10  c.c.  of  HC1  to  10  c.c.  of  urine  and  heat  the  mix- 
ture. The  presence  of  urochrome  is  indicated  by  a  bright 
red  color. 

Urea 

Place  a  drop  of  urine  on  a  slide,  add  a  drop  of  nitric 
acid,  and  partially  evaporate  by  warming  gently.  Crys- 
tals of  urea  nitrate  will  show  if  urea  is  present. 

Weyl's  Test  for  Creatinine 

To  5  c.c.  of  urine  add  10  drops  of  sodium  nitro- 
prusside  (saturated  solution).  Mix  and  add  10  drops  of 
potassium  hydroxide  (20%).  A  ruby-red  color  results 
which  soon  turns  yellow. 

To  the  yellow  solution  obtained  in  Weyl's  test  above 
add  an  excess  of  acetic  acid  and  apply  heat.  A  green 
color  results  and  is  in  turn  displaced  by  a  blue  color. 
If  creatinine  is  present,  a  precipitate  of  Prussian  blue  will 
form. 

Uric  Acid 

To  30  c.c.  of  urine  add  2  c.c.  HC1 ;  let  it  stand  24  hours. 
Crystals  of  uric  acid,  if  present,  will  separate  out. 

Murexid  Test 

Place  2  c.c.  of  urine  in  a  porcelain  dish,  add  I  drop  of 
nitric  acid,  and  evaporate  to  dryness.  Cool  and  add  I 
drop  of  ammonia.  A  blue  or  violet  color  indicates  the 
presence  of  uric  acid  or  urates. 


38. 


Clinical  Laboratory  Technic 


Urinary  Sediments 

The  urinary  sediment  is^tjie  deposit  which  is  found  in 
the  urine  after  standing.     This' sediment  is  classified  as 


<9b 


D 


URINE  CRYSTALS — Gibson 


i.  Triple  Phosphate.  2.  Calcium  Oxalate.  3.  Calcium  Car- 
bonate. 4.  Uric  Acid.  5.  (a)  Ammonium  Urate ;  (b)  Sodium 
Urate.  6.  Leucin.  7.  Cystin.  8.  Tyrosin.  9.  Cholesterin. 

non-organized,  or  chemic,  and  organized,  or  anatomic 
deposits. 

The  sediment  examination  is  useful  in  showing  the 
presence  of  abnormal  elements  excreted  by  the  kidneys. 

When  the  concentrated  sediment  has  been  obtained, 
either  by  centrifugalization  or  sedimentation,  a  drop  is 
taken  from  the  tube  with  a  pipette.  Place  the  index  finger 
on  the  upper  opening  of  the  pipette  and  insert  it  into  the 
tube,  carrying  it  down  into  the  fluid  to  the  sediment ;  then 


Urine  39 

release  the  pressure  of  the  finger  and  allow  a  few  drops 
to  enter  the  pipette,  maintaining  a  firm  pressure  of  the 
finger  on  the  upper  end  while  withdrawing  the  pipette. 
Place  a  drop  on  a  slide  and  examine  with  a  low  power 
lens  and  subdued  light,  then  with  high  power  and  slightly 
more  light. 

The  non-organized  sediments  are  usually  crystalline, 
although  a  few  are  amorphous.  The  crystalline  deposits 
are  important  only  when  found  in  freshly  voided  urine ; 
they  may  be  precipitated  in  any  urine  which  stands  and 
undergoes  fermentation. 

The  organic  sediments  consist  of  various  cells,  casts, 
yeast  fungi,  spermatozoa,  and  bacteria. 

Non-organized   Sediments  Occurring  in   Normal 
Urine 

URIC  ACID  CRYSTALS  :  Rhombic  prisms,  rosettes,  and 
hexagonal  plates,  usually  yellow — may  be  colorless.  Solu- 
ble when  heated  with  NaOH. 

CALCIUM  OXALATE  CRYSTALS:  Colorless,  envelope  and 
dumb-bell  shaped.  They  are  derived  from  various  foods. 

CALCIUM  CARBONATE  CRYSTALS  :  These  crystals  are 
found  in  alkaline  urine,  sometimes  in  slightly  acid  urine. 
It  crystallizes  in  the  form  of  granules,  spicules,  and 
dumb-bells. 

AMMONIUM  MAGNESIUM  PHOSPHATES:  "Triple  Phos- 
phates " ;  these  occur  in  sediment  in  two  forms,  the  pris- 
matic form  of  crystals  and  the  amorphous,  feathery  type. 
In  faintly  acid  urine  they  have  the  coffin-lid  appearance, 
but  in  alkaline  urine  they  take  a  variety  of  shapes.  These 
are  pathological  when  found  in  fresh  urine. 

AMORPHOUS  PHOSPHATES  of  Ca  and  Mg  are  commonly 


40  Clinical  Laboratory  Technic 

found  in  alkaline  urine.  They  appear  as  bulky,  opaque, 
white  deposits,  and  this  whitish,  flocculent  deposit  is 
precipitated  by  heat  and  dissolved  by  acetic  acid.  These 
earthy  phosphates  are  the  only  salts  of  phosphoric  acid 
that  are  found  in  the  urinary  sediment. 

AMMONIUM  URATES:  This  salt  of  uric  acid  is  formed 
during  ammoniacal  fermentation  of  the  urine,  and  is  only 
abnormal  if  the  urine  is  fresh ;  any  urine  upon  standing 
for  several  hours  is  apt  to  deposit  crystals  of  uric  acid. 
Acid  ammonium  urate  occurs  as  yellowish  red  or  dark 
brown  spherical  bodies,  radially  striated  and  studded  with 
fine,  prismatic  spicules — "thorn  apple  crystals." 

AMORPHOUS  URATES  are  of  little  importance.  They 
have  a  pinkish  or  brick-dust  appearance,  due  to  uroeryth- 
rin,  a  coloring  matter  found  in  the  urine  of  acute  rheu- 
matism. They  also  occur  as  rods  and  spikes ;  are  dissolved 
by  NaOH,  but  not  by  acetic  acid. 

Non-organized  Sediments  Occurring  in  Abnormal 
Urine 

CYSTIN  CRYSTALS  :  Colorless  hexagonal  plates  or  quad- 
rilateral prisms ;  soluble  in  HC1  and  ammonia,  insoluble 
in  acetic  acid,  water,  alcohol,  and  ether. 

LEUCIN  CRYSTALS  are  highly  refractive,  yellow  spheres, 
resembling  fat  globules ;  insoluble  in  ether. 

TYROSIN  CRYSTALS  appear  as  fine,  radiating  needles. 
They  are  colorless.  Tyrosin  and  leucin  crystals  are  found 
together  in  nephritis,  gout,  cirrhosis,  and  carcinoma  of  the 
liver. 

BILIRUBIN  AND  HEMATOiDiN  CRYSTALS  appear  as  red 
granules,  needles,  and  rhombic  plates.  They  have  no 
clinical  significance. 


Uric  Acid  Crystals. — From  Purdy  after  Peyer 


Ammonium    Urates,    showing    Spherules    and    Thorn-apple- 
shaped  Crystals. — From  Ogden  after  Peyer. 


Urine  41 

CRYSTALS  OF  URIC  ACID  may  be  suggestive  of  gravel 
or  calculus  if  blood  and  other  symptoms  are  present. 

The  presence  of  an  excessive  number  of  calcium  oxalate 
crystals  may  signify  oxaluria. 

AMMONIUM-MAGNESIUM  PHOSPHATE  CRYSTALS,  when 
formed  in  fresh  ammoniacal  urine,  are  suggestive  of  in- 
fection of  the  urinary  passages  and  calculus. 

CHOLESTERIN  CRYSTALS  are  large,  regular,  and  irregu- 
lar plates.  These  crystals  are  found  in  the  sediment  in 
cystitis,  nephritis,  pyelitis,  and  chyluria. 

Organized  Sediment 

Various  forms  of  epithelia  are  found  in  nearly  all 
urines.  They  are  the  formed  elements  coming  from  the 
different  parts  of  the  genito-urinary  tract,  and  are  sup- 
plied by  the  progressive  desquamation  of  the  mucous 
surfaces.  An  increase  in  number  indicates  inflammatory 
process  in  the  part  producing  them.  They  are  more  or  less 
granular  and  possess  one  or  more  nuclei.  The  size,  shape, 
and  condition  of  their  protoplasm  and  nuclei  should  be 
carefully  observed.  If  the  sediment  is  of  such  volume 
and  density  as  to  obscure  blood  cells,  casts,  and  pus,  dilute 
with  a  drop  of  clear  urine. 

Large,  round,  epithelial  cells,  with  dense  refractive 
protoplasm,  come  from  the  neck  of  the  bladder;  other 
large,  round  cells  may  come  from  the  membranous  and 
prostatic  urethra. 

Epithelium  from  the  Urinary  Tract 

Small,  round,  epithelial  cells  may  come  from  the  renal 
tubules,  the  pelvis  of  the  kidney,  or  the  urethra.  They 
are  mononuclear  and  slightly  larger  than  the  leucocytes. 


42  Clinical  Laboratory  Technic 

Large,  polygonal,  squamous  cells  come  from  the  bladder, 
but  they  may  also  enter  the  urine  from  the  prepuce  of  the 
male  or  vulva  of  the  vagina. 


5 

°00 


8 


9 


10 


EPITHELIUM  FROM  THE  URINARY  TRACT  —  Gibson 

I.  Squamous  epithelium  from  the  bladder.  2.  Cells  from  the 
neck  of  the  bladder.  3.  Epithelium  from  the  prostatic  urethra. 
4.  Cells  from  the  seminal  passages.  5.  Red  blood  cells.  6.  Leu- 
cocytes. 7.  Scaly  epithelium.  8.  Compound  granule  cell.  9.  Ure- 
thral  cells.  10.  Pelvic  cells. 

Cylindrical  and  caudate  cells  may  come  from  the  neck 
of  the  bladder  or  the  pelvis  of  the  kidney. 


Urine  43 

Leucocytes  are  present  in  small  numbers  in  normal 
urine.  An  increase  in  number  denotes  an  inflammatory 
condition  of  some  portion  of  the  urinary  tract.  They  are 
recognized  by  their  polymorphic  nuclei.  In  acid  urine 
they  are  often  shrunken,  and  in  alkaline  urine  they  are 
often  degenerated  and  swollen. 

Pus 

The  difference  between  a  pus  cell  and  a  leucocyte  is 
only  quantitative.  When  many  leucocytes  are  present,  the 
term  pus  is  used ;  when  few,  they  are  called  leucocytes. 

The  origin  of  pus  is  important.  Pus  from  the  kidney 
is  usually  mixed  with  casts  and  small,  round  cells ;  if  with 
squamous  cells,  it  is  probably  from  the  bladder.  A  cathe- 
terized  specimen  is  necessary  in  order  to  rule  out  the 
vagina  as  a  source. 

Red  blood  cells  are  pale,  non-granular,  non-nucleated 
discs.  In  concentrated  urine  they  may  present  a  crenated 
appearance,  and  in  alkaline  urine  they  are  often  destroyed, 
forming  masses  of  brown  granules.  Normal  blood  indi- 
cates a  hemorrhage  in  the  pelvis  or  bladder;  abnormal 
blood  signifies  that  it  is  higher  up  in  the  tubules. 

Casts 

Casts  are  cylindrical  formations  which  originate  in  the 
uriniferous  tubules ;  they  generally  indicate  some  kidney 
disorder.  They  have  uniform  marginal  outlines  with  well- 
defined  borders,  and  show  the  molded  effect  of  the  kidney 
tubules. 

HYALINE  CASTS  :  Transparent,  their  shape  and  char- 
acter may  be  determined  by  staining  with  iodine  solution, 
gentian  violet,  or  fuchsin.  Generally  albumin  is  present 
with  this  type  of  cast. 


44 


Clinical  Laboratory  Technic 


GRANULAR  CAST:  The  basic  substance  of  this  cast  is 
hyaline,  and  the  granules  consist  of  albumin,  degenerated 
epithelial  cells,  erythrocytes,  and  leucocytes.  They  are 


Hyaline . 


Granular. 


Epithelial. 


w 

\  _ 

Blood. 


Mucous   shreds. 


Waxy. 


CASTS  —  Gibson 

finely  granular  and  coarsely  granular,  according  to  the 
nature  and  size  of  the  granules. 

EPITHELIAL  CASTS:  Hyaline  casts  covered  with  epi- 
thelial cells  from  the  lining  of  the  uriniferous  tubules. 
These  are  particularly  abundant  in  acute  nephritis. 

BLOOD  CASTS  :  Hyaline  basis,  covered  with  erythrocytes. 
They  are  characteristic  of  acute  diffuse  nephritis  and 


Urine  45 

acute  congestion  of  the  kidney,  and  they  denote  renal 
hemorrhage. 

FATTY  CASTS  :  Fat  globules  and  fatty  acid  crystals  are 
deposited  upon  hyaline  or  granular  casts.  They  indicate 
fatty  degeneration  of  the  kidney,  and  are  found  in  sub- 
acute  and  chronic  inflammation  of  the  kidney. 

WAXY  CASTS:  Similar  to  the  hyaline  form,  but  are 
somewhat  larger  and  appear  more  solid,  having  a  sharper 
outline  and  a  light  yellow  color. 

CYLINDROIDS:  Flat  in  structure,  with  smaller  diameter 
than  casts,  having  branching  ends.  These  "false  casts" 
may  become  coated  with  granules  and  appear  granular 
in  structure. 

Mucous  SHREDS:  These  shreds  of  mucus  are  long, 
wavy,  transparent  bodies,  which  are  much  thinner  than 
casts  or  cylindroids. 

Tubercle  Bacilli  in  Urine.  (J.  A.  M.  A.,  March,  1915) 
Acidify  the  urine  with  30%  acetic  acid,  2%  of  its 
volume  with  a  $%  solution  of  tannic  acid.  Place  this  in 
the  ice  chest  24  hours.  Centrif  ugalize  the  precipitate,  then 
redissolve  with  a  dilute  acetic  acid  solution ;  centr  if  ugalize 
again,  and  smear  the  sediment  on  a  slide  and  stain.  The 
precipitate  may  be  treated  with  normal  sodium  hydroxide 
solution,  then  cultivated. 

Other  Cellular  Elements 

Spermatozoa,  cells  from  neoplasms,  micrococcus  ureas, 
streptococcus,  staphylococcus,  colon,  typhoid,  and  tubercle 
bacilli  may  find  entrance  and  grow  in  the  urinary  tract. 
To  be  of  diagnostic  value,  care  should  be  taken  to  pre- 
vent their  entering  from  other  sources. 


46  Clinical  Laboratory  Technic 

Examination  of  bacteria  should  be  upon  fresh  urine 
obtained  by  catheterization. 

Molds  and  yeasts  are  sometimes  found  in  diabetic  urine. 

Animal  parasites,  booklets,  and  daughter  cysts  of  echi- 
nococcus,  and  bilharzia  hematobia  ova  occasionally  find 
their  way  into  the  urine. 


v 


OTHER  CELLULAR  ELEMENTS  OF  URINE — Gibson 

i.  Spermatozoa.  2.  Micrococcus  urese.  3.  Molds.  4.  Bacilli. 
5.  Vinegar  eel.  6.  Yeast  fungi.  7.  Bilharzia  hematobia  eggs. 
8.  Echinococcus  hooklets. 

Burnham's  Test  for  Formaldehyde 

To  10  c.c.  of  urine  in  a  test  tube  slightly  warmed,  add 
3  drops  of  5%  alcoholic  phenylhydrazine  solution,  one 
small  crystal  of  nitro-prusside. 

Overlay  with  2  c.c.  of  a  saturated  solution  of  NaOH. 

The  excretion  of  formaldehyde  by  the  kidneys  of 
patients  taking  urotropin  varies,  as  the  kidneys  of  some 
patients  do  not  decompose  the  drug,  and  urotropin 
depends  for  its  action  on  formaldehyde,  into  which  it 
is  decomposed  in  the  kidney.  Positive  reaction1  will 

1  If  the  test  is  negative  it  shows  that  the  urotropin  is  not  being  broken 
up  in  the  body,  and  is  doing  the  patient  no  good. 


Urine  47 

become   deep  purplish  black,  changing  quickly  to   dark 
green,  gradually  getting  lighter. 

Systematic  Scheme  in  Charting 

The  following  terms  are  commonly  used  in  qualitative 
reactions : 

MACROSCOPICAL — Slightest  possible  trace  ;  faint  trace ; 
trace;  small  amount;  moderate  amount;  large  amount; 
very  large  amount. 

MICROSCOPICAL — An  occasional ;  a  few ;  a  moderate 
number ;  many ;  very  many. 

Quantitative  Estimation  of  Phosphates 

Place  50  c.c.  of  urine  in  a  flask  and  add  5  c.c.  of  sodium 
acetate  solution.  Heat  the  mixture  to  boiling  point.  A 
standard  solution  of  uranium  nitrate  is  run  into  the  hot 
mixture  by  means  of  a  burette,  until  a  precipitate  ceases 
to  form  and  a  drop  of  the  mixture  assumes  a  brownish 
red  color  when  brought  in  contact  with  a  drop  of  potas- 
sium ferrocyanide  solution  in  a  porcelain  dish. 

This  is  the  end  point  of  the  precipitation. 

The  number  of  c.c.  of  uranium  solution  used  is  read  off 
from  the  scale  on  the  burette. 

i  c.c.  of  the  standard  uranium  solution  is  equivalent  to 
0.005  gram  of  P2O5  (phosphoric  acid).  Therefore  the 
number  of  grams  of  P2O5  in  50  c.c.  of  urine  is  estimated 
by  multiplying  by  0.005. 

EXAMPLE.  10  c.c.  of  uranium  solution  were  used. 
Then  in  50  c.c.  of  urine  there  are  10  X  0.005,  or  0.05; 
and  in  100  c.c.  of  urine  there  are  10  X  0.005  X  2,  or 
0.1%  P2O,. 

Normally,  3.5  grams  per  day. 


48  Clinical  Laboratory  Technic 

Quantitative  Estimation  of  Chlorides.     (Volhard- 
Harvey  Method) 

Pipette  5  c.c.  of  urine  into  a  porcelain  dish  and  dilute 
with  20  c.c.  of  distilled  water. 

Precipitate  the  chlorides  with  exactly  10  c.c.  of  standard 
silver  nitrate  solution;  add  2  c.c.  of  ferric  ammonium 
sulphate  (indicator). 

A  solution  of  standard  ammonium  sulphocyanate  is  run 
in  from  a  burette  until  a  yellowish  color  appears  in  the 
mixture. 

Subtract  the  number  of  c.c.  of  sulphocyanate  solution 
used  from  10  c.c.,  the  quantity  of  silver  nitrate  solution 
taken,  and  this  will  give  the  number  of  c.c.  of  the  silver 
nitrate  solution  actually  used  in  the  precipitation  of  the 
chlorides. 

I  c.c.  of  silver  nitrate  solution  is  equivalent  to  o.oi  gram 
of  sodium  chloride,  and  the  number  of  c.c.  of  silver  nitrate 
solution  used  multiplied  by  o.oi  gram  will  give  the  weight 
of  sodium  chloride  in  5  c.c.  of  urine.  Calculate  from  this 
the  weight  of  sodium  chloride  in  24-hour  amount  of  urine. 

The  weight  of  chlorine  may  be  estimated  by  multiply- 
ing by  the  factor  0.006. 

Total  Acidity.     (Folin's  Method.) 

Use  a  preservative  in  the  twenty- four  hour  amount  of 
urine  in  order  to  avoid  decomposition.  Place  25  c.c.  of 
urine  in  an  Erlenmeyer  flask  (250  c.c.  capacity).  Add  15 
grams  of  finely  powdered  potassium  oxalate  and  2  drops 
of  i%  phenolphthalein  solution.  Shake  vigorously  for 
one  minute  and  immediately  titrate  with  N/io  sodium 
hydroxide  until  a  faint  but  permanent  pink  color  ap- 
pears. Note  the  number  of  cubic  centimeters  of  the 
N/io  sodium  hydroxide  used  and  calculate  the  acidity. 


Urine  49 

EXAMPLE.  6.5  c.c.  of  N/io  sodium  hydroxide  were 
used;  then:  25  16.5  ::  1,500 :x.  25x  equals  9,750. 

x  equals  390  (acidity  of  24-hour  urine  expressed  in 
cubic  centimeters  of  N/io  sodium  hydroxide). 

Each  cubic  centimeter  of  N/io  sodium  hydroxide  con- 
tains 0.004  gram  of  sodium  hydroxide,  and  this  is  equiva- 
lent to  0.00^3  gram  of  oxalic  acid.  To  express  the  total 
acidity  of  the  24-hour  amount  in  equivalent  grams  of 
sodium  hydroxide,  multiply  x  by  0.004.  To  express  the 
total  acidity  in  grams  of  oxalic  acid,  multiply  x  by  0.0063. 

EXAMPLE.  390  times  0.004  equals  1.560  grams  of 
sodium  hydroxide  in  24-hour  amount.  390  times  0.0063 
equals  2.4570  grams  of  oxalic  acid  in  24-hour  amount. 

It  is  considered  more  difficult  to  increase  than  decrease 
the  acidity  of  the  urine.  The  acidity  of  the  urine  may 
be  increased  after  the  administration  of  mineral  acids, 
acid  phosphates,  or  benzoates ;  in  cardio-renal,  and  other 
disorders,  in  fasting,  and  in  acidosis. 

Test  for  Ammonia 

Pipette  25  c.c.  of  fresh  urine  into  an  Erlenmeyer  flask 
of  250  c.c.  capacity.  Add  5  c.c.  of  a  saturated  solution 
of  potassium  oxalate  that  has  been  neutralized  to  phe- 
nolphthalein,  then  add  3  drops  of  a  i%  alcoholic  solution 
of  phenolphthalein.  Run  in  from  a  graduated  burette 
N/io  sodium  hydroxide  until  a  faint  pink  color  appears. 
Then  add  5  c.c.  of  40%  commercial  formalin  that  has 
been  neutralized  to  phenolphthalein  and  again  titrate  to 
the  same  color. 


50  Clinical  Laboratory  Technic 

Each  cubic  centimeter  of  N/io  sodium  hydroxide  used 
in  this  last  titration  equals  i  c.c.  of  N/no  ammonia,  or 
0.0017  gram  of  ammonia. 

Multiply  0.0017  by  the  number  of  cubic  centimeters 
used  in  the  last  titration.  This  gives  the  number  of 
grams  of  ammonia  in  25  c.c.  of  urine. 

Calculate  the  quantity  of  ammonia  in  the  twenty- four 
hour  amount  of  urine. 

The  amount  of  ammonia  in  the  urine  varies  with  the 
amount  of  protein  ingested.  One  or  two  grams  in 
twenty- four  hours  is  considered  a  normal  amount ;  more 
than  this  amount  indicates  an  acidosis. 

Renal  Functional  Test.    (Rowntree  and  Gerahty, 
Journal  A.  M.  A.,  1911) 

METHOD.  The  bladder  should  be  emptied,  then  give 
the  patient  500  c.c.  of  water. 

Exactly  i  c.c.  of  the  phenolsulphonephthalem  solution 
from  an  ampoule,  which  contains  more  than  i  c.c.,  is  in- 
jected into  the  lumbar  muscles.  In  10  minutes  obtain  a 
specimen  and  add  a  few  drops  of  alkali ;  then  at  the  end 
of  an  hour  from  the  time  the  specimen,  rendered  alkaline, 
first-  shows  coloration,  the  entire  contents  of  the  bladder 
should  be  carefully  collected  and  the  amount  of  drug 
excreted  accurately  estimated.  When  used  as  a  differen- 
tial test,  the  secretions  of  the  two  kidneys  should  be 
separately  collected  by  ureter  catheterization  for  i  hour 
from  the  time  the  urine  from  either  side  first  shows 
coloration. 

To  open  the  ampoule,  file  the  neck  between  the  small 
bulb  and  the  body  of  the  ampoule. 

Fill  the  wedge-shaped  cell  of  the  colorimeter  with  a 


Urine  51 

standard  solution  made  by  diluting  exactly  I  c.c.  of 
phenolsulphonephthalein  solution  from  an  ampoule  with 
200  c.c.  of  water,  adding  10  c.c.  of  a  5%  solution  of 
sodium  hydroxide,  and  then  sufficient  water  to  make 
i  liter. 

At  the  end  of  the  hour  from  the  time  the  specimen, 
rendered  alkaline,  first  shows  coloration,  collect  the  urine 
for  2  hours,  each  hour's  specimen  being  kept  in  separate 
bottles,  labelled  first  hour  and  second  hour. 

Dilute  the  specimen  of  urine  with  200  c.c.  of  water  and 
render  alkaline  with  10  c.c.  of  $%  solution  of  sodium 
hydroxide,  then  further  dilute  to  make  i  liter.  Fill  the 
rectangular  cup  to  the  mark.  The  cup  is  then  placed  in 
the  apparatus  and  the  latter  manipulated  until  the  colors, 
as  seen  through  the  prism,  are  identical,  when  the  per- 
centage of  excretion  will  be  indicated  on  the  scale.  If 
the  coloration  is  slight,  showing  small  excretion  of  the 
phthalein,  then  the  dilution  should  be  carried  only  to 
250  or  500  c.c.  and  the  readings  on  the  scale  divided  by 
4  or  2. 


EXAMPLE.    First  hour 

Second  hour         27% 


Total  65%   (normal) 

REFERENCES:  Physiological  Chemistry,  Hammarsten. 
Text-Book  of  Physiology,  Howell.  Clinical  Examination 
of  Urine,  Ogden.  Physiological  Chemistry,  Hawk.  Grad- 
wohl's  Blood  and  Urine  Chemistry. 


Clinical  Laboratory  Technic 


Breakfast 


10.00  A.M. 


11.30  A.M. 


2.00  P.M. 


4.30   P.M. 


8  to  10  P.M. 


Acute  Nephritic  Diet 

Milk,  6  oz. 

Cream  (top  cream),  3  oz. 
Zwieback  (one  slice,  3x2x1) 
i  cubic  inch  butter 

Milk,  6  oz. 
Cream,  2  oz. 
Sugar,  i  oz. 

Milk,  6  oz. 
Cream,  2  oz. 
Zwieback  (ii  slices) 
Butter  (i  cubic  inch) 

Milk,  6  oz. 
Cream,  2  oz. 
Sugar,  i  dr. 

Milk,  6  oz. 
Cream,  2  oz. 
Rice  (3  tb.) 
Sugar,  4  dr. 
Zwieback  (i  slice) 
Butter 


Calories 


Once  at  night,  if  awake 


Milk,  6  oz. 
Cream,  2  oz. 

Milk,  6  oz. 
Cream,  2  oz. 


2,900 


Courtesy  of  The  Boston  City  Hospital. 


Low  Salt  Diet  ("Salt  Free") 

One  liter — 32  oz.  of  milk  in  24  hours. 
Salt  Free  Bread  )  ad  Ub 
Salt  Free  Butter  ) 

Sugar,  rice,  potatoes,  stewed  fruit,  fresh  green  vegetables,  choco- 
late, coffee,  tea,  cereals,  salad  without  salt. 
Small  portions  of  meat. 
One  or  two  eggs  daily. 

Add  no  salt  to  food 


Urine 


S3 


Diet  in  Nephritis 


Breakfast.  (Any  one  food  from 
each  group  may  be  given) 

1.  Cereals : 

.Cooked  4tb.. 

Cornflakes  8  tb. 

Shredded  wheat  I  biscuit 

2.  Meat : 

Lamb  chop  I 

Pork  chop  i 

Mutton  chop  i 

Fish  2  tb. 

Oysters  8 

3-  Eggs  I 

4.  Bread  I  slice 

Muffins  I 

Rolls  i 

Supper.    (Any  one  food  from 
each  group  may  be  given) 

1.  Cereals : 

Cornflakes  8  tb. 

Cooked  4  tb. 

2.  Vegetables : 

Green  corn  3  tb. 

Peas  2  tb. 

Prunes  8  tb. 

3.  Oysters  8 

4-  Eggs  i 

5.  Bread  i  slice 

6.  Coffee  or  tea,  with 

milk  and  sugar. 


Dinner.    (Any  one  food   from 
groups  i,  2,  4,  5,  6;  any 

two  from  group  3) 

1.  Soup  32  oz. 

2.  Meats: 

Steak  (2  x  i  x  i  in.) 
Chops  (lean)  i 

Fish  2  tb. 

3.  Vegetables.       (Any  two    of 

these  may  be  given) 
Potato  2  tb. 

Mashed  4  tb. 

Parsnips  4  tb. 

Carrots  4  tb. 

Squash  4  tb. 

Turnips  4  tb. 

Onions  4  tb. 
Butter  beans  2tb. 

Lima  beans  2  tb. 

4.  Macaroni  4tb. 
Vermicelli  4  tb. 

5.  Puddings : 

Rice  4  tb. 

Tapioca  2  tb. 

Bread  2  tb. 

Cornstarch  2  tb. 

Ice  cream  2  tb. 

6.  Bread  i  slice 

Rolls  i 

Muffins  i 

The  following  may  be  given 
ad  lib.:  Butter,  olive  oil,  olives, 
tomatoes,  lettuce,  celery,  sweet 
fruits  of  any  kind,  sugar. 

Courtesy  of  The  Boston  City  Hospital. 


54 


Clinical  Laboratory  Technic 


Strict  Diabetic  Diet 


Meats. 

Beef,  mutton,  ham,  bacon, 
poultry,  shrimp,  bologna, 
sausage,  lamb,  pork  chops, 
steak,  tongue,  pigs'  feet, 
brains,  bone  marrow,  smoked 
or  pickled  meats,  scraped  or 
corned  beef. 

Fish. 

All  kinds.  No  dressing  con- 
taining flour.  Crabs,  lobsters, 
sardines,  etc. 

Soups. 

Clear  (not  containing  a  fari- 
naceous substance).  Beef 
juice. 

Gelatin. 

Eggs. 

Prepared  any  way,  with  large 
amounts  of  butter. 

Butter, olive  oil:  large  amounts. 
Cheese. 

French  dressing  (olive  oil,  vin- 
egar, etc.). 

Coffee,  tea,  without  sugar. 
Akoll  and  Alpha  biscuits. 


Vegetables,  etc.  ,with  5%  or  less. 
Lettuce,  spinach,  string  beans, 
celery,  asparagus,  cucumbers, 
Brussels  sprouts,  unspiced 
pickles,  olives,  grape  fruit, 
cauliflower,  tomatoes,  rhu- 
barb, clams,  scallops. 

Foods,  6%  or  less. 

Cabbage,  radishes,  pumpkin, 
oysters,  liver. 

Foods,  10%  or  less. 

Onions,  squash,  turnip,  car- 
rots, beets,  lemons,  oranges, 
cranberries,  peaches. 

100  G.    Carbohydrate  Diet 
Strict  diet  o. 

Vegetables  (5%,  6%,  10% 

groups)  10. 

Cream,  ?  pt.  6. 

Oatmeal,  I  gill  (dry)     24. 
Bread,  i  oz.  30. 

Potato,  i  medium,  baked 

or  mashed;  2  tb.          15. 
Orange  or  grape  fruit, 


loogms. 
Courtesy  of  The  Boston  City  Hospital. 


Urine  55 


Urine 
Name Age Sex Date. 

Color. 

Odor. 

Sediment. 

Specific  Gravity. 

Reaction. 

Albumin. 

Sugar. 

Bile. 

Indican. 

Microscopical  Examination. — • 

SPECIAL  CHEMICAL  EXAMINATION 
24°  amount. 
Blood. 
Acetone. 
Diacetic  acid. 
/3-oxybutyric  acid. 
Sugar — quantitative. 
Albumin — quantitative. 
Total  solids. 
Chlorides. 
Total  nitrogen. 
Ammonia. 
Creatinine. 
Urea. 
Uric  acid. 
Phosphates. 

Microscopical  Examination. — 

Signed 


CHAPTER  IV 
FECES 

The  Feces  are  the  residue  which  remains  after  com- 
plete digestion  and  absorption  in  the  intestines,  and  the 
residue  is  different  qualitatively  and  quantitatively  accord- 
ing to  the  variety  and  quantity  of  food.  The  normal  stool 
varies  in  quantity  from  250  to  500  grams. 


FIG.  47.  —  MICROSCOPICAL  CONSTITUENTS  OF  FECES.  (v.  Jaksch.) 
a,  Muscle  fibers ;  b,  connective  tissue ;  c,  epithelium ;  d,  leuco- 
cytes;  e,  spiral  cells;  f,  g,  h,  i,  various  vegetable  cells;  k,  "triple 
phosphate"  crystals;  I,  woody  vegetable  cells;  the  whole  inter- 
spersed with  innumerable  micro-organisms  of  various  kinds. — 
From  Stiffs  "Practical  Bacteriology.11 

Color 

The  color  of  normal  feces  is  due  to  hydrobilirubin, 
also  called  stercobilin,  and  it  originates  from  the  bilirubin 
which  is  secreted  into  the  intestine  in  the  bile,  being 
formed  by  the  reducing  activity  of  certain  bacteria. 

A  mixed  diet  produces  a  stool  which  varies  in  color 

56 


Feces  57 

from  light  to  dark  brown ;  meat  diet,  a  dark  stool ;  milk 
diet,  a  light  stool. 

Calomel  colors  the  stool  green;  bismuth  subnitrate, 
black;  senna  and  rhubarb,  yellow;  iron  gives  a  gray  or 
black  stool. 

Blood  will  color  the  stool  black  if  the  source  is  in  the 
alimentary  canal.  Greenish  yellow,  liquid  stools  are  char- 
acteristic of  typhoid.  Sprue  stools  are  whitish,  putty 
colored,  filled  with  air  bubbles. 

Consistency 

Normally,  the  consistency  may  vary  from  a  well- formed 
stool  to  a  pasty  discharge. 

The  feces  are  soft  when  the  absorption  is  prevented, 
intestinal  secretion  increased,  as  in  cholera,  and  where 
there  is  an  increase  of  fat.  In  steatorrhea  the  stools  are 
pale  and  greasy. 

Constipation  gives  a  hard,  compacted  stool,  also  scyba- 
lous  masses.  When  there  is  a  stricture  of  the  lower  bowel, 
the  feces  occur  in  slender,  cylindrical  masses. 

Odor 

Due  to  skatole  and  indole,  two  gaseous  products  of 
proteid  decomposition,  and  also  to  hydrogen  sulphide. 

Reaction 

Normally  alkaline  to  slightly  acid. 

Micro-chemical  Examination  of  Feces 

Thoroughly  mix  feces,  transfer  a  portion  as  large  as  a 
walnut  to  a  mortar,  and  grind  with  water  to  a  thin  mush. 
Place  2  drops  on  two  separate  slides. 


58  Clinical  Laboratory  Technic 

The  first  drop  is  simply  covered  with  a  cover  glass  and 
examined  for  the  following:  muscle  fibers,  connective 
tissue,  fatty  acids,  soaps,  leucocytes,  erythrocytes,  vege- 
table cells,  micro-organisms. 

The  second  drop  is  mixed  with  a  drop  of  36%  acetic 
acid  and  heated  until  bubbles  appear.  Cover  with  a  glass 
and  examine  for  fatty  acid  flakes.  This  also  differentiates 
between  connective  tissue  and  mucus,  the  former  being 
rendered  transparent. 

Add  2  drops  of  Sudan  III  or  Scharlach  R.  to  the  third 
drop.  This  will  stain  the  fat  globules  red. 

A  drop  of  Lugol's  solution  is  added  to  the  fourth  drop, 
as  this  will  stain  starch  granules,  fungi,  yeasts,  etc.,  blue 
or  violet. 

Food 

Shreds  of  undigested  food  may  be  due  to  improper 
mastication. 

.In  a  meat  diet  the  muscle  fibers  show  their  characteristic 
striations. 

Fat  and  Fatty  Acid 

When  fat  is  taken  too  freely  into  the  diet,  and  in 
diseases  of  the  pancreas,  fat  appears  in  the  stools  as  highly 
refractive  globules.  It  is  soluble  in  ether. 

Soaps 

Irregular  yellow  masses  of  fat. 

Mucus 

Mucus  gives  the  stool  a  glairy,  slimy  appearance.  If 
mixed  with  fecal  material,  it  is  from  the  small  intestine ; 
otherwise  it  is  from  the  colon. 


Feces  59 

Carbohydrates 

These  appear  in  single  starch  granules  and  in  masses 
of  granules  in  cellulose  envelopes. 

Guaiac  Test  for  Blood 

To  10  c.c.  of  fecal  suspension  add  5  drops  of  acetic 
acid;  shake.  Extract  with  10  c.c.  of  ether  and  decant 
5  c.c.  Add  12  drops  of  freshly  prepared  gum  guaiac 
solution  (take  a  piece  the  size  of  a  pea  and  dissolve  in 
70%  alcohol),  then  add  20. to  30  drops  of  hydrogen  per- 
oxide. A  bluish  color  will  result  if  blood  is  present. 

Dry  Test  for  Occult  Blood.     (Journal  A.  M.  A., 

January  30,   1915) 

Place  a  clean  slide  on  a  sheet  of  white  paper  and  smear 
with  feces.  Cover  with  benzidin  reagent.  Blue  or  green 
color  indicates  blood. 

Benzidin  Reagent 

Benzidin  I  gram 

Glacial  acetic  acid  2  c.c. 

H2O2  (3%)  20  drops 

Benzidin  Test  for  Occult  Blood 

Make  a  thin  fecal  suspension  with  distilled  water,  and 
heat  to  boiling  to  render  the  oxidizing  enzymes  inactive. 
To  2  c.c.  of  saturated  solution  of  benzidin  in  glacial  acetic 
acid  add  3  c.c.  of  3%  hydrogen  peroxide  and  2  to  3  drops 
of  the  cooled  fecal  suspension. 

A  clear  green  or  blue  color  appears  within  I  to  2 
minutes  in  the  presence  of  blood. 

If  the  mixture  is  not  shaken,  a  ring  of  color  will  form 
at  the  top.  This  test  is  of  value  only  in  cases  of  organic 
disease,  and  has  for  its  condition  a  diet  without  meat. 


60  Clinical  Laboratory  Technic 

Phenolphthalein  Test 

To  4  c.c.  of  thin  fecal  suspension  add  2  c.c.  of  the 
phenolphthalein  reagent  (i  to  2  grams  of  phenolphthalein 
and  25  grams  of  KOH  in  100  c.c.  of  distilled  water.  Add 
10  grams  of  powdered  zinc  and  heat  gently  until  the 
solution  is  decolorized).  Add  a  few  drops  of  hydrogen 
peroxide.  A  pink  or  red  color  promptly  forms  in  the 
presence  of  blood. 

Hydrobilirubin 

Mix  a  small  amount  of  feces  with  a  few  c.c.  of  concen- 
trated mercuric  chloride  (HgCl2),  and  allow  to  stand 
6  to  24  hours.  If  hydrobilirubin  is  present,  it  will  be  indi- 
cated by  a  deep  red  color,  and  this  is  due  to  the  forma- 
tion of  hydrobilirubin  mercury.  If  unaltered  bilirubin  is 
present  in  any  portion  of  the  feces,  that  portion  will  be 
green  in  color,  due  to  the  oxidation  of  bilirubin  to  bili- 
verdin. 

Bilirubin 

r  This  is  normally  reduced  to  hydrobilirubin,  and  is  not 
found  in  the  feces,  as  a  rule,  unless  this  process  is  inter- 
fered with,  as  by  too  rapid  passage  of  the  intestinal  con- 
tents through  the  canal.  The  absence  of  both  bilirubin 
and  hydrobilirubin  has  an  important  bearing  upon  the 
diseases  of  the  bile  passages. 

Gmelin's  Test  for  Bile 

Place  a  few  drops  of  concentrated  nitric  acid  in  an 
evaporating  dish,  and  allow  a  few  drops  of  feces  and 
water  to  mix  with  it.  The  usual  play  of  colors — green, 
blue,  violet,  red,  and  yellow — is  produced  if  bile  is 
present. 


Feces  61 

Pus 

It  is  difficult  to  detect  pus  in  the  feces  unless  there  is  a 
large  amount.  Mix  a  small  amount  of  feces  with  normal 
salt  solution,  which  is  isotonic,  and  make  a  smear.  Stain 
with  Wright's  stain. 

Test  for  Trypsin 

Make  a  casein  solution  by  dissolving  0.5  gram  of  casein 
(which  must  be  chemically  pure:  white,  not  yellowish) 
in  500  c.c.  of  a  1-1,000  solution  of  sodium  carbonate. 
Test  this  solution  by  adding  i%  acetic  acid.  If  a  precipi- 
tate forms,  the  solution  is  all  right. 

In  a  series  of  six  tubes  place  5  c.c.  of  the  casein  solution. 

Make  a  fecal  suspension  by  dissolving  I  gram  of  feces 
in  100  c.c.  of  a  1-1,000  sodium  carbonate  solution.    Clear 
by  filtering.    Tube  6  used  as  control. 
To  tube  number  i  add  i       c.c.  of  the  fecal  suspension. 

({  ((  (C  _  U  ^£  ((  {(  ((  ((  <( 

«         it  (f  ((      _.  £ 


It       <(       « 


<(         «  a        -       ((     ~  T        ((       «       «         «  " 

"      "  "     6    "    o.o     "     "     "      " 

Add  a  few  drops  of  toluol  to  each  tube,  shake,  let  stand 
at  40°  C.  for  24  hours.  Then  add  to  each  tube  a  few 
drops  of  acetic  acid  (i%)  solution.  If  casein  is  present, 
there  is  a  precipitate.  If  it  has  been  digested  (by  trypsin), 
there  is  no  precipitate. 

Test  for  Diastase 

In  each  tube  of  a  series  of  six  place  2  c.c.  of  a  yV% 
solution  of  soluble  starch.  As  a  control  for  reagent  add 
iodine  solution  to  tube  6,  to  see  if  the  starch  is  all  right. 

Make  a  thin  fecal  suspension  in  sodium  carbonate 
1-10,000  (i  gram  of  feces  to  100  c.c.  of  solution). 


62  Clinical  Laboratory  Technic 

To  tube  number  i  add  i       c.c.  of  the  fecal  suspension. 

"         "  «         2       fi          >rr      "        "      "          "  " 

ft         «  «         ~       «   •  ~  -         a        a      ((          n  (( 

O  ^O 

(t  ((  ((  .  t(  g         ((  U          ((  II  (( 

"     "  "      5     "   o.i      "     "    "      " 

"     "  "      6    "   o.o     "     "    "      " 

Incubate  30  minutes  at  40°  C.,  then  add  to  each  tube  a 
few  drops  of  iodine  or  Lugol's  solution.  If  the  starch 
is  undigested  the  reaction  is  a  deep  blue  color,  showing 
pancreatic  insufficiency. 

Micro-organisms 

Various  species  of  micro-organisms  are  found  in  the 
feces.  Streptococci  fecalis  are  found  in  normal  stools — 
a  non-virulent  type.  About  one-third  of  the  dry  substance 
of  stools  of  a  normal  adult  consists  of  bacteria.  B.  coli. 
B.  lactis  aerogenes,  and  B.  Welchii  are  the  gas-forming 
organisms.  The  fecal  output  of  bacteria  decreases  under 
the  influence  of  water-drinking  with  meals. 

Tubercle  Bacilli 

In  intestinal  tuberculosis  the  bacilli  are  found  imbedded 
in  the  mucus.  They  may  have  their  origin  from  sputum 
which  has  been  swallowed. 

Take  a  small  amount  of  the  stool  and  mix  with  water; 
centrifugalize.  Place  a  drop  of  the  scum  on  a  slide,  dry, 
stain  with  carbol-fuchsin  stain  (Ziehl-Neelsen). 

Various  vegetable  detritus  may  be  mistaken  for  in- 
testinal parasites,  as  banana,  which  simulates  tapeworm 
segment ;  and  celery  fibers,  oxyuris.  The  oxyuris  vermi- 
cularis  (pinworm)  is  often  found  in  the  stools  of  children. 


Feccs 


Important  pathogenic  protozoa  of  the  intestinal  tract.  (ia)  Mo- 
tile E.  coli.  Note  large  amount  and  peripheral  arrangement  of 
chromatin  in  nucleus,  (ib)  Encysted  E.  coli.  Note  larger  size 
than  E.  histolytica  cyst,  8  ring  form  nuclei  and  absence  of  chromi- 
dial  bodies.  (2)  Motile  E.  histolytica  from  acute  dysenteric 
stool.  Note  histolytica  nucleus  with  scanty  chromatin.  (3)  Tet- 
ragena  type  of  E.  histolytica  from  case  of  chronic  dysentery. 
Note  greater  amount  of  chromatin  and  central  karyosome  with 
centriole.  (40)  Preencysted  E.  histolytica  from  carrier.  Note 
small  size  and  heavy  peripheral  ring  of  chromatin  in  nucleus 
making  this  feature  of  chromatin  in  nucleus  similar  to  the  larger 
E.  coli.  (4b)  Encysted  E.  histolytica  from  dysentery  convales- 
cent. Note  small  size,  4  ring  nuclei  and  a  dark  chromatin  stain- 
ing mass,  "chromidial  body."  (5a  and  5&)  Motile  and  encysted 
cultural  amoebae  from  Manila  water  supply.  (6a  and  6b)  Oocyst 
and  sporozoite  production  in  4  spores  of  Eimeria  stiedce.  (ja  and 
7&)  Oocyst  with  2  sporoblasts  and  oocyst  with  2  spores  contain- 
ing 4  sporozoites  of  Isopora  bigemina.  (8a  and  8fr)  Vegetative 
and  encysted  Trichomonas  intestinalis.  (ga  and  gb)  Vegetative 
and  encysted  Lamblia  intestinalis.  (10)  Balantidium  eoli.  (Illus- 
trations of  amoeba  from  Walker — others  from  Doflcin.} — From 
Stiffs  "Practical  Bacteriology." 


64 


Clinical  Laboratory  Technic 


Examination  of  Feces  for  Eggs.   (Journal  A.  M.  A.) 

1.  Thoroughly  mix  2  grams  of  feces  with  5  c.c.  of  a 
2%  solution  of  cresol  in  a  centrifuge  tube. 

2.  Centrifugalize  at  high  speed  I  minute,  then  decant 
and  add   fresh  cresol  solution,  mix,  and  centrifugalize 
again.     Repeat  the  process  three  times. 


Ova  of  the  Parasitic  Worms  or  Man 
TREMATODA 

.  o    ft 

Heterophyes 
heteroplwes 


.OA     AWN  TO  SCALCX16OO 


lancetxtum' 


Fasciola 


Clnorciiis  Clonorchis 


Schistqsoma 


soiotx 


istosoma 


—From  Stitt's  "Practical  Bacteriology." 

3.    Remove   a  small  portion  of  the  bottom  sediment 

with  a  clean  pipette  and  place  on  a  slide  ;  add  a  drop  of 

anilin  gentian  violet,  mix,  and  place  a  cover  glass  over 

it.    The  entire  slide,  except  the  real  eggs;  is  stained  violet. 


Ova  of  the  Parasitic  Worms  of  Man 
NEMATODA 

OR  AWN  TO         SC.At.E.          X         IOOO 

^^5TiAA 

&£|®  SSSSgr . 

rf/  I  *&Sj?p&  1       convex  rj 

M         ^^w    w 

Oxyuris 

xnfocus^BrSurface  focus      \^py  '  VermfculOTiS  A.B. 

is.wo5  /  I         V__^/  /^~X 

Strongylus     /JP^ 
subtilis    \Kfh. 

'.-Without  outer       fifcHH  NeCOtOT 

envelope  (Modified  Wl^y  americanus    *s 

^tltfef*2 '  &Ud       I  fC^i  y   (M<^fi*d  trt^SMe*.  W*         /| 

.^SOT-L  ^-^4/ 

^p  A0chylostorna  /  AgchylostoniS 

H^^^   duodenale          Embryo/       "JP8Sen^e 
A.  B:  CD.  ;     Trichuris    ^-^      ^  ^rJngyToid^ 

trlchiura  ^jteicor^^  • 

CESTOD^"" 


D.-Atypical,  unfertilized 


I - 

C4 


Ttenia  IWn,         fl"  """ 

solium  DiolcK^^  Dibothno^—       M         -*r 

^onoporas    cephalus      Hymenpjepisna^ 

v  ^  ...^nolepis 
^  %  diminuta 


is 


J^P1       ^ 
Teenia 
saginata^ 

*  Dipylidium  "^S 

Cestode  sedments 

VwUVt!  r*£~\  DRAWN    TO    SCAU£    X  »O       O 


m 


^£SPensis  Hymeno- 
/  \    lepls 

V^^       V!i/  I          dinrinuta 

pS?A  ^'808?  &.      -  \         (Modified  fc-wa*tSle*J9< 

Tbenia  Dipylidium 
solium  caninum    Dibothriocei*ialus  latus 


66  Clinical  Laboratory  Technic 

Infants'  Stools 

The  examination  of  infants'  stools  is  very  important 
and  a  great  aid  in  determining  the  nature  of  the  disturb- 
ances of  digestion  in  infancy.  The  technic  for  micro- 
scopical examination  is  the  same  as  for  adult  stools 
(see  page  57).  As  nearly  all  dusting  powders  used  on 
babies'  buttocks  contain  starch,  this  fact  must  be  kept 
in  mind  when  testing  with  iodine  solution. 

Meconium 

The  meconium  is  the  fecal  matter  discharged  by  the 
newborn.  Bile-tinged  mucoid  intestinal  secretions  fol- 
low meconium,  then  the  normal  fecal  stool. 

The  starvation  stool  resembles  the  meconium.  It  is 
a  small  brownish-green  stool,  composed  of  bile,  intestinal 
secretions,  and  bacteria. 

Number 

The  number  of  stools  varies.  Four  in  24  hours  is  con- 
sidered normal.  Fermentative  and  infectious  processes 
increase  the  number.  As  a  rule,  breast-fed  babies  have 
more  evacuations  than  bottle-fed,  due  to  the  high  sugar 
percentage  of  breast  milk. 

Form 

Usually  soft  and  unformed.  Scybalous  and  crumbly 
stools  are  due  to  an  increased  soap  content.  Foamy 
stools  are  due  to  fermentation  in  the  intestine,  usually 
of  carbohydrates,  sometimes  of  proteins. 

Color 

Normally,  golden-yellow  in  color. 

The  most  common  abnormal  color  is  green,  and  this  is 
generally  due  to  the  change  of  bilirubin  to  biliverdin. 


Feces  67 

This  color  may  be  due  to  excessive  acidity  or  alkalinity 

or  to  the  action  of  the  bacillus  pyocyaneous. 

White 

These  stools  are  composed  chiefly  of  unabsorbed  fat  in 
the  form  of  soaps. 

Gray 

As  a  rule  gray  stools  are  due  to  absence  of  bile,  and  the 
presence  of  some  form  of  fat,  usually  soap.  Bile  pig- 
ment may  be  present  in  the  form  of  the  colorless  leuco- 
hydrobilirubin.  (Test  for  hydrobilirubin,  page  60.) 

Black 

The  black  stool  is  usually  due  to  the  action  of  some 
drug,  such  as  bismuth,  iron,  or  charcoal.     In  rare  in- 
stances it  is  due  to  changed  blood. 
Reaction 

Normally,  slightly  acid,  neutral,  or  slightly  alkaline. 

Strongly  acid  stool  generally  due  to  fermentation  of 
carbohydrates. 

Abnormal  Constituents 
Blood 

May  be  due  to  inflammation  of  the  large  intestine, 
intussusception,  crack  in  the  anus,  or  infectious  diarrhea. 

Pus 

Pus  is  present  in  inflammation  of  the  large  intestine. 
It  is  found  microscopically  in  most  cases  of  inflammation 
of  the  colon. 

Mucus 

Never  present  macroscopically  in  normal  stools.  It 
occurs  in  infectious  diarrhea,  and  is  present  in  large 
amounts  in  fermentative  diarrhea. 


68  Clinical  Laboratory  Technic 

Curds 

Casein  curds  are  firm  and  tough  and  are  insoluble  in 
ether. 

The  fat  curds  are  smaller,  easily  broken  up,  and  are 
soluble  in  ether  after  acidification  and  heating. 

REFERENCES:  Parasitologie  Animale,  Neveu-Lemaire. 
Physiological  Chemistry,  Hammarsten.  Morse  and  Talbot. 

Fat  Free  Diet 

Breakfast.  Calories 

Skimmed  milk       I  glass  (6  oz.)  84 

Toasted  bread      2  slices  120 

Boiled  egg  i  80    284 

Dinner. 

I.    Meats      2  chops  (lamb  or  pork). 

Must  be  lean. 

or          Steak  (2x2x2)  200 

Chicken 

Roasts  (beef,  lamb),  lean 
II.      Fish        i  portion 

III.  Fresh  vegetables    Lettuce 

Spinach  50 

Peas 

String  beans 

IV.  Toasted  bread      2  slices  120 

V.     Skimmed  milk       I  glass  (6  oz.)  80  450 

or  or 

VI.    Light  soups  (without  vegetables)  6  oz.      20  390 

Supper. 

Cooked  fruits  Prunes  or  apricots                            80 

1  boiled  egg  80 

2  slices  toast  120 

i  glass  skimmed  milk  84    364 

Give  no  fats,  greasy  foods,  sugar,  butter,  nor  whole  milk. 

Courtesy  of  The  Boston  City  Hospital. 


Feces  69 


Feces 

Name Spec.. 

Date. 


Quantity  Test  Diet  Feces 

Character  Quantity 

Macroscopic  Exam.  Character 

Mucus  Macroscopic  Exam. 
Parasites 

Microscopic  Exam.  Microscopic  Exam. 
Pus  Proteid 

Blood  Carbohydrates 

Parasites  Fats 

Blood  (guaiac  test)  Fermentation  test 

Hydrobilirubin 

REMARKS  : 

Signed 


CHAPTER  V 
GASTRIC  CONTENTS 

Vomitus 

All  vomitus  should  be  carefully  examined  and  the 
amount,  color,  odor,  and  consistency  noted.  Tests  for 
free  hydrochloric  acid  may  be  made,  but  it  is  seldom  used 
for  chemical  analysis.  Microscopic  examination  should 
be  made  for  necrotic  shreds,  red  blood  cells,  and  sarcinae. 

Gastric  Contents  , 

The  fractional  method  of  gastric  analysis  is  consid- 
ered the  most  accurate  method  of  examining  gastric  con- 
tents. Ewald's  test  meal  is  given,  then  at  intervals  of 
15  minutes  from  the  time  the  meal  is  given,  until  the 
stomach  is  empty,  5  to  6  c.c.  are  withdrawn  from  the 
stomach  by  the  Rehfuss  tube.  Normally  30  to  50  c.c. 

The  acidity  of  the  gastric  contents  is  determined  by 
titration  with  a  standard  alkali,  using  the  indicator  to 
determine  the  end  point  of  the  titration. 

The  true  acidity  of  a  solution,  that  is,  its  hydrogen  ion 
concentration,1  cannot  be  determined  always  by  titration. 

Character 

Normal  contents  are  thin  and  watery,  sometimes  bile- 
colored,  frothy,  and  may  contain  mucus  and  cells. 

Abnormal  contents  may  be  red  from  blood.  Changed 
blood  has  the  appearance  of  "coffee  grounds." 

xFor  u  Methods  of  Determining  Hydrogen  Ion  Concentrations" 
see  "Determination  of  Hydrogen  Ions/'  by  Clark. 

70 


Gastric  Contents  yi 

A  few  starch  granules  or  fat  droplets  may  be  found  in 
normal  contents,  but  more  than  this  indicates  stasis. 

Under  normal  conditions  the  gastric  contents  become 
acid  15  minutes  after  the  ingestion  of  food,  and  this  is 
due  to  the  presence  of  free  acid  or  acid  salts,  lactic  acid, 
and  traces  of  hydrochloric  acid.  Within  an  hour  hydro- 
chloric acid  predominates  and  no  lactic  acid  is  found. 
The  contents  should  be  examined  as  soon  as  possible,  as 
lactic  acid  readily  develops  if  the  contents  are  left  in  a 
warm  place. 

Hyper-acidity  is  an  excessive  degree  of  acidity. 

Hypo-acidity  is  a  deficiency  of  acid. 

Hyper-chlorhydria  is  a  large  amount  of  hydrochloric 
acid  secreted  by  the  stomach  cells. 

Hypo-chlorhydria  is  a  small  amount  of  hydrochloric 
acid. 

Achlorhydria  is  the  absence  of  hydrochloric  acid. 

Achylia  Gastrica  is  the  absence  of  ferments  of  the 
gastric  juice. 

Litmus  indicates  the  presence  or  absence  of  acid 
elements. 

The  presence  of  free  hydrochloric  acid  shows  that  the 
acid  secreting  power  of  the  stomach  is  not  destroyed. 
Its  absence  in  fasting  contents  has  no  significance. 

Congo  Red  Paper  Test 

Moisten  the  end  of  the  congo  red  paper  with  the  gastric 
contents,  and  if  hydrochloric  acid  is  present  the  red  is 
changed  to  blue. 

Topfer's  Test 

(Topfer's  reagent  is  a  0.5%  alcoholic  solution  of 
dimethyl-amido-azo-benzene. ) 


72  Clinical  Laboratory  Technic 

To  10  c.c.  of  gastric  contents  add  2  drops  of  Topfer's 
reagent.  A  carmine  color  indicates  the  presence  of  a  free 
mineral  acid. 

Mucin 

Filter  5  c.c.  of  contents,  add  a  few  drops  of  acetic  acid. 
A  white  precipitate  indicates  mucin. 

Gastric  Contents 

Congo  Red  Test  for  HC1 

i  c.c.  of  gastric  contents,  10  c.c.  of  water,  and  2  drops 
of  a  concentrated  solution  of  congo  red  in  50%  alcohol. 
The  presence  of  acid  is  indicated  by  a  blue  color. 

Resorcin  Test 

Dissolve  3  grams  of  cane  sugar  and  5  grams  of  resorcin 
in  100  c.c.  of  alcohol. 

Equal  drops  of  this  reagent  and  gastric  contents  are 
slowly  evaporated  to  dryness  in  a  porcelain  dish.  A  rose 
red  color  indicates  the  presence  of  HC1. 

Uffelmann's  Test  for  Lactic  Acid 

Place  2  drops  of  carbolic  acid  and  6  drops  of  ferric 
chloride  solution  (U.  S.  P.)  in  a  test  tube  and  add  water 
until  the  solution  is  a  deep  amethyst  blue.  Add  5  drops 
of  gastric  contents.  The  presence  of  lactic  acid  will  be 
indicated  by  a  lemon-yellow  color. 

Gunzburg's  Test  for  Free  Hydrochloric  Acid 

Place  a  drop  of  the  contents  in  a  porcelain  dish  and 
gently  warm.  As  soon  as  it  begins  to  evaporate  add  a 
drop  of  Gunzburg's  reagent  so  it  will  just  touch  the  other 


Gastric  Contents  73 

drop;  heat  without  burning.    Red  crystals  appear  at  the 
zone  of  contact  if  free  mineral  acid  is  present. 

Ferric  Chloride  Test  for  Lactic  Acid 

To  10  c.c.  of  gastric  contents  add  10  c.c.  of  ether,  shake, 
then  decant  the  ether  extract. 

Dilute  the  ferric  chloride  solution  until  the  yellow  color 
is  almost  lost,  and  to  this  add  the  ether  extract.  The  zone 
of  contact  becomes  yellow  if  lactic  acid  is  present. 

Tests  for  Food 

Filter  10  c.c.  of  contents  and  add  I  drop  of  osmic  acid. 
Fat  droplets  will  stain  black;  a  drop  of  Sudan  III  will 
stain  the  droplets  red. 

Add  i  drop  of  tincture  of  iodine  to  10  c.c.  of  filtered 
contents.  Starch  granules,  if  present,  will  appear  blue. 

Test  for  Pepsin 

Add  sufficient  HC1  to  give  the  test  for  free  HC1.  Boil 
an  egg  5  minutes  and  place  the  coagulated  white  in  a  tube. 
Place  the  tube  open  end  down  in  the  gastric  contents  and 
incubate  |  to  4  hours.  The  albumin  will  show  signs  of 
digestion  if  pepsin  is  present. 

Test  for  Rennin 

Neutralize  10  c.c.  of  gastric  contents  with  NaOH,  add 
10  c.c.  of  milk,  and  incubate  10  to  15  minutes.  The  milk 
will  coagulate  if  rennin  is  present. 

Guaiac  Test  for  Blood 

To  15  c.c.  of  gastric  contents  add  5  drops  of  glacial 
acetic  acid,  and  shake.  Extract  with  10  c.c.  of  ether  and 
decant  5  c.c.  Add  12  drops  of  gum  guaiac  solution  (a 


74  Clinical  Laboratory  Technic 

piece  of  guaiac  the  size  of  a  pea  dissolved  in  70%  alcohol), 
freshly  prepared.  Add  20  to  30  drops  of  hydrogen  perox- 
ide. A  bluish  color  will  result  if  blood  is  present. 

Benzidin  Test  for  Occult  Blood 

Boil  the  gastric  contents,  then  cool.  Add  2  c.c.  of  a 
saturated  solution  of  benzidin  in  acetic  acid  to  10  c.c.  of 
the  cooled  gastric  contents.  Shake,  and  add  30  drops 
of  hydrogen  peroxide.  A  bluish  green  color  is  positive. 

When  there  is  but  small  amount  of  material  to  test  for 
acidity,  use 

N 

—  NaOH  for  every  100  c.c.  of  material. 

i 

—  NaOH     "        "         10  c.c.    " 

10 

—  NaOH     "       "          5  c.c.   " 

20 

Tests  for  Common  Poisons 

Reinsch's  Test  for  Arsenic 

Acidify  the  vomitus  or  stomach  contents  with  HC1, 
and  allow  to  stand  i  hour.  Filter  through  several  layers 
of  gauze.  Immerse  a  brightly  polished  strip  of  copper 
foil  in  the  filtrate  and  boil.  A  gray  coating  will  appear 
on  the  copper  if  arsenic  is  present.  Dissolve  this  in  nitric 
acid  and  evaporate  to  dryness.  Add  a  few  drops  of  silver 
nitrate  to  the  residue.  A  red  precipitate  indicates  the 
presence  of  arsenite  of  silver. 

Carbolic  Acid 

Acidify  the  gastric  contents  with  HC1,  filter,  and  add 
a  few  drops  of  bromine  water.  Carbolic  acid  gives  a 
yellow  precipitate. 


Gastric  Contents  75 

Chloral 

To  10  c.c.  of  gastric  contents  add  10  c.c.  of  ether. 
Shake,  and  decant  the  ether;  add  i  c.c.  of  silver  nitrate 
and  i  c.c.  of  ammonia.  Boil  3  minutes.  In  the  presence 
of  chloral  a  mirror  of  metallic  silver  should  form. 

Corrosive  Sublimate 

Add  10  c.c.  of  ether  to  10  c.c.  of  gastric  contents;  mix 
thoroughly.  Decant  the  ether  and  concentrate  it  by 
evaporation.  Add  potassium  iodide.  The  presence  of 
mercury  is  indicated  by  a  precipitate  of  iodide  of  mercury, 
and  this  is  soluble  in  potassium  iodide. 

Hydrochloric  Acid 

To  10  c.c.  of  filtered  gastric  contents  add  a  few  drops 
of  nitric  acid  and  2  c.c.  of  silver  nitrate  solution.  Silver 
chloride  appears  as  a  white  precipitate.  As  normal  gastric 
contents  contains  HC1,  make  a  control  with  0.2%  HC1 
solution  and  compare  the  density  of  the  two  precipitates. 

Nitric  Acid 

The  contents  are  neutralized  with  carbonate  of  potash. 
Filter  and  concentrate  the  filtrate  by  evaporation.  Mix 
with  an  equal  amount  of  sulphuric  acid.  Cool,  then  add 
a  solution  of  ferrous  sulphate.  Nitric  acid  is  indicated  by 
the  appearance  of  a  black  ring  at  the  junction  of  the  two 
fluids. 

Oxalic  Acid 

Add  caustic  potash  to  the  contents  and  boil.  Potassium 
oxalate  will  form  even  if  lime  has  been  given  to  the  patient. 
Filter  and  add  calcium  sulphate  solution.  A  white  pre- 
cipitate indicates  oxalate,  and  this  is  soluble  in  HC1, 
insoluble  in  acetic  acid. 


76  Clinical  Laboratory  Technic 

Phosphorus 

To  10  c.c.  of  contents  add  10  c.c.  of  water;  acidify  with 
sulphuric  acid.  Place  this  in  a  glass  retort  with  a  long 
condensing  tube  and  distil  in  the  dark.  The  condensing 
vapors  are  made  luminous  by  the  traces  of  phosphorus. 

Prussic  Acid 

Neutralize  the  contents  and  distil  slowly  on  a  water 
bath.  Add  silver  nitrate.  Prussic  acid  gives  a  white 
precipitate,  soluble  in  hot  nitric  acid,  insoluble  in  cold. 
Prussian  blue  will  form  by  adding  liquor  potassae,  hydro- 
chloric acid,  and  a  few  drops  of  sulphate  of  iron. 

Sulphuric  Acid 

Add  a  few  drops  of  barium  chloride  to  the  filtered 
contents.  A  white  precipitate  indicates  sulphuric  acid, 
which  is  insoluble  in  HC1  or  HNO3. 

Alkaloids. — Otto's  Method 

Acidify  the  contents  with  dilute  sulphuric  acid  to  con- 
vert the  alkaloid  into  sulphate.  Filter  through  several 
layers  of  gauze.  Mix  with  ether  to  remove  fat.  Pour  off 
the  ether  and  add  small  amounts  of  potassium  carbonate 
to  liberate  the  alkaloid.  Mix  with  ether  again,  pour  off 
the  ether,  and  test  as  follows : 

Belladonna 

To  5  c.c.  of  the  ethereal  solution  add  a  few  drops  of 
nitric  acid  and  evaporate  upon  a  water  bath.  Cool  and 
add  i  drop  of  potash  dissolved  in  absolute  alcohol.  The 
presence  of  atropin  is  indicated  by  the  appearance  of  a 
violet  color  changing  to  red. 


Gastric  Contents  77 

Opium 

Dissolve  5  c.c.  of  the  ethereal  solution  which  has  been 
evaporated  to  dryness  with  cold  sulphuric  acid.  Add 
i  drop  of  20%  bichromate  of  potash.  A  bright  green 
color  indicates  the  presence  of  morphine. 

Strychnine 

Add  2  drops  of  sulphuric  acid  to  the  ethereal  solution 
which  has  been  evaporated  to  dryness,  and  2  drops  of 
20%  bichromate  of  potash.  Strychnine  will  give  a  purple 
blue  color  changing  to  red. 

Examination  of  Fasting  Contents.     (12  hours'  fast) 

Amount. — Normally,  10  to  12  c.c. 

Character. — Clear,  sediment,  sirupy,  ropy. 

Food  Residue. — Macroscopic:  Normal  or  excess  of 
food,  sediment.  Microscopic :  Epithelial  cells,  blood,  bac- 
teria, sarcinae  (indicative  of  stasis). 

Blood. — Guaiac  Test. 

Reaction. — Litmus,  phenolphthalein. 

Free  HC1. — Topfer's  reagent. 

Lactic  Acid. — Fe2Cl6,  ethereal  extract. 

Mucin. — Acetic  acid. 

Ewald's  Test  Meal 

Wheat  bread,  35  to  70  grams. 

Water,  300  to  400  c.c. 

Given  when  the  stomach  is  empty,  i.  e.,  as  a  breakfast. 

Examination  of  Contents 

Quantity.  Over  50  to  60  c.c.  is  abnormal,  indicating 
hypersecretion  or  stasis.  500  or  more  suggests  dila- 
tion of  stomach,  benign  or  malignant  stenosis  of  the 


78  Clinical  Laboratory  Tech  me 

pylorus.  If  well  digested,  there  should  be  a  layer  of  finely 
divided  bread  residue  on  the  bottom  of  the  glass  contain- 
ing the  stomach  contents,  and  over  this  a  layer  of  semi- 
transparent  gastric  juice.  If  poorly  digested,  the  contents 
will  consist  of  fluid  and  many  coarse  lumps  of  bread. 

Character. — The  proportion  of  solids  to  liquids  is 
i  to  3.  The  proportion  of  liquids  is  much  higher  in 
hypersecretion. 

Color. — Blood  or  bile. 

Odor. — Normal  (faintly  sour)  or  fetid  (rancid). 

Reaction. — Litmus.  HC1  is  decreased  in  many  chronic 
cases :  carcinoma  of  stomach  and  advanced  gastritis ;  it  is 
often  absent  in  neurasthenic  and  hysterical  individuals. 
The  absence  of  HC1  affords  favorable  opportunity  for 
bacterial  action  in  the  stomach,  and  lactic  acid  is  a  very 
common  product  of  this  bacterial  fermentation.  Large 
amounts  of  lactic  acid  are  suggestive  of  carcinoma,  and 
is  generally  accompanied  by  the  Boas-Oppler  bacillus, 
which  plays  an  important  part  in  the  lactic  fermentation. 
This  organism,  which  is  identical  with  the  Bulgarian 
bacillus,  is  found  in  75  to  85%  of  the  cases  of  carcinoma 
of  the  stomach. 

Enzymes. — Pepsin,  rennin.  The  absence  of  pepsin  and 
rennin  is  less  common  than  that  of  HC1.  The  estimation 
of  pepsin  is  chiefly  of  value  in  cases  suggesting  an  ad- 
vanced lesion  of  gastric  mucosa,  and  in  which  free  HC1 
has  been  known  to  be  absent. 
Quantitative  Test  for  Free  Acidity. — Fractional  Method 

Sahli's  reagent  is  used,  as  it  is  considered  more  ac- 
curate than  Topfer's.1 

1Sahlfs  reagent:  a  mixture  of  equal  parts  of  a  48%  solution  of 
potassium  iodide  and  an  8%  solution  of  potassium  iodate. 


Gastric  Contents  79 

i  c.c.  of  strained  gastric  contents  is  measured  with  an 
Ostwald  pipette  and  placed  in  a  60  c.c.  porcelain  dish. 
Add  10  c.c.  distilled  water  and  I  c.c.  of  Sahli's  reagent. 
Let  stand  five  minutes,  then  titrate  with  N/ioo  sodium 
thiosulphate  until  a  faint  yellow  color  remains.  Add  5 
to  10  drops  of  a  i%  solution  of  soluble  starch.  Titrate 
until  blue  color  disappears. 

Calculation 

N/ioo  thiosulphate  is  equivalent  to  N/ioo  alkali ;  this 
value  indicates  the  number  of  cubic  centimeters  of  N/ioo 
sodium  hydroxide  necessary  to  neutralize  the  free  hydro- 
chloric acid  in  i  c.c.  gastric  contents.  Note  the  number 
of  cubic  centimeters  of  N/ioo  thiosulphate  required  to 
titrate  i  c.c.  gastric  contents  to  the  total  disappearance  of 
blue  color  in  the  presence  of  starch.  Multiply  by  10  to 
obtain  the  number  of  cubic  centimeters  of  N/io  NaOH 
necessary  to  neutralize  100  c.c.  gastric  contents. 

Quantitative    Test    for    Total    Acidity. — Fractional 
Method 

Add  i  c.c.  of  strained  gastric  contents  to  15  c.c.  of 
distilled  water,  and  2  drops  of  a  i%  alcoholic  solution  of 
phenolphthalein.  Titrate  with  N/ioo  sodium  hydroxide 
until  a  faint  pink  color  is  obtained. 

Calculation 

Multiply  the  number  of  cubic  centimeters  of  N/ioo 
NaOH  required  to  neutralize  i  c.c.  of  gastric  contents  by 
10  to  obtain  the  number  of  cubic  centimeters  N/io  NaOH 
necessary  to  neutralize  100  c.c.  of  gastric  contents. 

To  express  the  acidity  of  100  c.c.  gastric  contents  in 
terms  of  hydrochloric  acid,  by  weight,  multiply  the  value 
obtained  by  0.00365.  i  c.c.  of  N/io  hydrochloric  acid 
contains  0.00365  gram  of  hydrochloric  acid. 


8o  Clinical  Laboratory  Technic 

Tryptophane  Test.     (A.  M.  A.,  LV,  1910) 

Four  or  five  hours  after  a  regular  meal  some  contents 
are  secured  and  filtered.  Mix  5  c.c.  of  the  juice  with 
sterile  2%  Witte's  peptone,  add  i  c.c.  of  toluene,  shake, 
and  incubate  at  body  temperature  24  hours.  At  the  end 
of  that  time  3  or  4  c.c.  of  the  mixture  are  taken  and,  if 
not  acid,  treated  with  a  few  drops  of  3%  acetic  acid.  Add 
bromine  water  (saturated  solution),  drop  by  drop,  until 
in  case  the  reaction  is  positive,  a  reddish  violet  color 
appears. 

This  test  is  based  on  the  fact  that  carcinomatous  tissue 
contains  an  enzyme  of  stronger  proteolytic  power  than 
pepsin,  causing  the  appearance  of  amino  acids,  including 
tryptophane. 

Blood  and  trypsin  must  not  be  present.  (The  latter 
may  be  regarded  as  absent  if  bile  has  not  been  detected.) 

Quantitative  Analysis  of  Gastric  Contents 

Topfer's  Method  for  determining  total  acidity,  com- 
bined acidity,  and  free  acidity,  also  acidity  due  to  organic 
acids  and  acid  salts. 

Total  Acidity 

Filter  10  c.c.  of  gastric  contents  and  add  3  drops  of 
i%  alcoholic  solution  of  phenolphthalein  (i  gram  of 
phenolphthalein  in  100  c.c.  95%  alcohol). 

Fill  the  burette  with  N/io  NaOH  and  take  a  reading. 
Titrate  until  a  pink  color  appears  which  does  not  dis- 
appear on  stirring  and  is  not  intensified  by  further  addi- 
tion of  a  drop.  The  free  HC1  is  then  neutralized  in  the 
contents  used. 

Take  a  reading  of  the  burette.  The  number  of  c.c. 
used  in  titrating  multiplied  by  0.00365  equals  the  acidity 
of  the  10  c.c.  of  the  gastric  contents  in  terms  of  HC1 


Gastric  Contents  81 

(i  c.c.  of  N/io  HC1  acid  contains  0.00365  gram  HC1 
acid). 

EXAMPLE.  "io  c.c.  of  the  filtered  gastric  contents  are 
neutralized  by  8.1  c.c.  of  N/io  NaOH  solution.  As  i  c.c. 
of  N/io  NaOH  neutralizes  0.00365  gram  of  HC1,  8.1  c.c. 
will  neutralize  0.00365  X  8.1  c.c.,  or  .0295  gram  of  HC1, 
the  amount  in  the  10  c.c.  of  gastric  contents;  and  in 
loo  c.c.  it  would  take  ten  times  as  much,  or  0.295  gram 
HC1  or  per  cent.1 

Normally,  the  percentage  is  0.07  to  0.18%  (or  20  to  50 
c.c.  N/io  NaOH  to  100  c.c.  gastric  contents). 

Combined  Acidity 

Add  3  drops  of  i%  of  an  aqueous  solution  of  sodium 
alizarin  sulphonate  to  10  c.c.  of  gastric  contents.  Take 
a  reading  of  the  burette.  Titrate  with  N/io  NaOH 
until  a  violet  color  appears.  Take  a  reading.  This  indi- 
cator used  reacts  to  all  acidities ;  therefore,  in  order  to 
determine  the  amount  of  N/io  NaOH  necessary  to  neu- 
tralize the  combined  acidity  of  10  c.c.  of  gastric  contents, 
subtract  the  burette  reading  from  the  reading  obtained 
in  the  determination  of  the  total  acidity. 

Free  Hydrochloric  Acid 

Filter  10  c.c.  of  gastric  contents  and  add  4  drops  of 
di-methyl-amino-azobenzol.  Take  a  reading  of  the 
burette.  Titrate  with  N/io  NaOH  until  the  red  color 
is  replaced  by  lemon-yellow.  If  the  lemon-yellow  appears 
as  soon  as  the  indicator  is  added,  free  acid  is  absent.  This 
indicator  reacts  only  to  free  acid,  therefore  the  amount 

Calculated  in  per  cent,  i.e.,  the  amount  in  100  c.c.  =  0.295 
per  cent 


82  Clinical  Laboratory  Technic 

of   N/io   NaOH   used   indicates   the  cubic   centimeters 
necessary  to  neutralize  10  c.c.  of  gastric  contents. 

Acidity  Due  to  Organic  Acids  and  Acid  Salts 

Subtract  the  number  of  c.c.  of  N/io  NaOH  used  to 
neutralize  the  free  acid  in  10  c.c.  of  gastric  contents  from 
the  number  of  c.c.  used  in  neutralizing  the  combined  acids. 
The  remainder  indicates  the  number  of  c.c.  of  N/io 
NaOH  necessary  to  neutralize  the  organic  acids  and  acid 
salts  in  10  c.c.  of  gastric  contents. 

Tryptophane  Test. — Second  Method 

1.  Test  meal — water  sweetened  and  bread.    Remove 
in  about  i  hour. 

2.  Incubate  part,  or  let  stand  at  room  temperature 
24  to  48  hours. 

3.  Filter  contents — 6  to  7  c.c.  in  a  test  tube. 

4.  Add  a  few  drops  of  3%  acetic  acid. 

5.  Add  saturated  aqueous  solution  of  bromine,  drop 
by  drop,  up  to  4  drops  from  a  pipette. 

RESULT.  Reddish  violet  or  rose  color  shows  trypto- 
phane.  If  the  color  does  not  come,  let  stand  15  to  20 
minutes.  If  negative,  add  more  bromine  solution,  drop 
by  drop,  until  mixture  is  yellowish  or  rose  red.  Let  stand 
and  reddish  color  grows  deeper.  Shake  after  each  addi- 
tional bromine. 

REFERENCES  :  Hawk's  Physiological  Chemistry ;  Ham- 
marsten's  Physiological  Chemistry;  Journal  of  the  A.  M. 
A.,  LV,  1910. 


Name. 


Gastric  Contents 

Stomach  Contents 

Spec.. 


Fasting  Contents 

Quantity 

Character 

Food  Residue 

Sediment 

Mucin 

Blood 

Reaction 

Free  HC1 

Lactic  Acid 

MICROSCOPICAL  EXAMINATION  : 


Date 

Test  Meal  Contents 

Quantity 

Character 

Reaction 

Free  HC1 

Total  Free  HC1 

Total  Acidity 

Combined  HC1 

Pepsin 

Rennin 


Signed. 


CHAPTER  VI 
SPUTUM 

The  Sputum  consists  of  saliva,  buccal  mucus,  and  the 
secretions  from  the  respiratory  passages. 

Quantity 

The  amount  expectorated  in  twenty-four  hours  varies. 
It  is  very  large  in  advanced  tuberculosis  with  cavity  for- 
mation, and  scanty  in  phthisis  and  bronchitis. 

Consistency 

Ordinarily-,  sputum  is  sticky,  viscous  fluid.  It  may, 
however,  be  purulent,  serous,  or  bloody. 

Muco-purulent  is  the  most  common  and  is  often  seen 
in  pneumonia,  bronchitis,  and  phthisis.  It  is  not  character- 
istic of  any  particular  condition. 

The  sputum  in  phthisis,  abscess  of  lung,  or  empyema  is 
purulent,  thick,  green,  or  yellow. 

Coin-like  masses  of  pus,  which  sink  in  water,  occur  in 
the  sputum  in  tuberculosis. 

Dittrich's  plugs  are  gray  or  yellowish  masses,  foul  smell- 
ing, and  are  characteristic  of  septic  bronchitis  and  gan- 
grene of  the  lung. 

Curschmann's  spirals  are  yellowish  coiled  masses  found 
in  the  sputum  of  bronchial  asthma. 

Color 

Black  or  gray  sputum  is  from  carbon  inhaled  or  due  to 
food, 


Sputum  85 

Rusty-colored  sputum  is  common  in  pneumonia. 

Greenish  discoloration  of  the  sputum  is  due  to  the 
activity  of  certain  chromogenic  bacteria. 

Sputum  discolored  with  streaks  of  blood  may  be  due 
to  coughing  or  vomiting. 

Odor 

Sputum  is  nearly  odorless.  If  it  has  stagnated  in  the 
body  it  may  have  a  putrid  odor.  On  standing  it  may 
acquire  a  foul  odor  from  decomposition.  In  tuberculosis 
it  has  a  sweet,  penetrating  odor. 

Reaction 

Fresh  sputum  is  generally  alkaline ;  on  standing  it  may 
become  acid. 

Microscopic  Examination 

The  unimportant  constituents  are  various  kinds  of 
epithelial  cells,  leucocytes,  sometimes  red  blood  cells,  parti- 
cles of  food,  and  fungi. 

The  important  elements  are  pus,  blood,  elastic  tissue, 
animal  parasites,  and  plant  parasites. 

A  specimen  should  be  stained  and  examined  for  bacteria 
associated  with  special  diseases,  as  tuberculosis,  pneu- 
monia, etc. 

Bacteria  in  Sputum.     (Smith's  Modification  of  Gram's 
Stain) 

1.  Make  a  smear,  dry,  and  fix  by  passing  through  a 
flame.     Cover    with    aniline-gentian    violet    10    seconds, 
gently  steaming.    Wash  in  water. 

2.  Gram's  iodine  solution  30  seconds.    Wash  with  95% 
alcohol  until  color  ceases  to  come  out. 


86  Clinical  Laboratory  Technic 

3.  Wash  with  ether  (to  remove  fat).    Absolute  alcohol 
5  seconds. 

4.  Saturated  aqueous  solution  of  eosin  2  minutes. 

5.  Wash  with  Loffler's  methylene  blue,  then  after  the 
excess  of  eosin  has  been  removed,  steam  with  methylene 
blue  5  seconds. 

6.  Wash  with  water,  rinse  with  alcohol,  and  clear  with 
xylol. 


§ 

0 

FIG.  20. — MICROSCOPICAL  CONSTITUENTS  OF  SALIVA 
#,  Epithelial  cells;  3,  salivary  corpuscles;  c,  fat  drops  ;  d,  leucocytes-, 
t,',    fj   and    gt    bacteria;    ^,    z,    and   k,    fission-fungi.  —  From  Haivk^s 
"Physiological  Chemistry" 

The  pneumococcus  is  stained  blue-black,  while  the 
capsule  is  stained  pink.  Influenza  bacilli  and  other  bac- 
teria which  do  not  stain  by  Gram's  method  are  clearly 
brought  out,  also  eosinophilic  leucocytes. 

The  tubercle  bacilli  are  slender,  non-motile  rods, 
occurring  singly  and  in  pairs,  usually  slightly  curved. 
Branching  and  club-shaped  forms  are  sometimes  ob- 
served, also  spherical  granules.  The  bacilli  have  a  thin 
capsule  which  contains  the  greater  portion  of  the  waxlike 
substance  peculiar  to  the  bacillus.  Spread  the  material 
in  a  thin  layer,  dry,  and  fix  by  passing  the  slide  through 
a  flame. 


Sputum  87 

Ziehl-Neelsen   Method  of  Staining  Tubercle  Bacilli 

1.  Cover  surface  of  the  slide  with  carbol-fuchsin  solu- 
tion, steaming  for  3  minutes  over  a  Bunsen  flame. 

2.  Wash  in  water. 

3.  Decolorize  with  20%   sulphuric  acid  until  the  red 
color  disappears. 

4.  Wash  thoroughly  in  water. 

5.  Wash  in  95%  alcohol  for  30  seconds. 

6.  Wash  in  water. 

7.  Stain  with  Loffler's  or  Gabbet's  stain.    The  tubercle 
bacilli  are  stained  red. 

There  are  many  other  acid- fast  bacilli  which  have  the 
same  staining  reactions  as  the  tubercle  bacilli,  but  they 
differ  in  having  no  pathogenic  power  for  guinea  pigs 
and  in  growing  readily  on  any  media. 

The  tubercle  bacillus  is  pathogenic,  not  only  to  man, 
but  also  to  animals. 

The  guinea  pigs  are  very  susceptible  and  are  much  used 
for  the  detection  of  tubercle  bacilli  in  suspected  material. 
An  intraperitoneal  injection  of  a  large  dose  will  cause 
death  in  10  to  20  days. 
« 

The  Determination  of  Types  of  Pneumococcus 

Several  types  of  pneumococcus  may  be  differentiated 
as  the  infectious  agent  in  lobar  pneumonia  with  immune 
sera. 

A  small  portion  of  sputum  which  is  known  to  be  rich 
in  pneumococci  is  placed  in  a  sterile  Petri  glass  dish 
and  washed  in  sterile  salt  solution.  It  is  then  ground 
up  in  a  sterile  mortar  with  i  c.c.  of  sterile  bouillon  salt 
solution,  i  c.c.  of  this  mixture  is  injected  into  the  peri- 
toneal cavity  of  a  white  mouse. 


88  Clinical  Laboratory  Tcchnic 

When  the  mouse  appears  sick,  the  belly  is  punctured 
with  a  capillary  pipette  and  a  smear  preparation  of  a 
drop  of  the  peritoneal  fluid  is  examined.  Repeat  the  pro- 
cedure if  the  fluid  be  not  rich  in  pneumococci. 

When  numerous  pneumococci  have  been  found,  kill 
the  mouse,  open  the  peritoneal  cavity,  and  wash  out, 
under  sterile  precautions,  with  5  c.c.  of  salt  solution, 
using  a  sterile  pipette  with  a  rubber  bulb.  Transfer  the 
washings  to  a  sterile  tube  and  centrifuge  at  low  speed 
for  a  short  time  to  throw  down  cells  and  fibrin. 

Draw  off  the  supernatant  fluid  and  centrifuge  at  high 
speed  until  the  organisms  are  deposited  as  sediment. 
Remove  the  supernatant  fluid  and  make  a  suspension 
of  the  same  concentration  as  that  of  a  bouillon  culture 
of  pneumococcus,  eighteen  hours  old. 

Set  up  five  small  tubes.  Introduce  0.5  c.c.  of  the  sus- 
pension in  the  first  four  tubes,  and  0.4  c.c.  in  the  fifth 
tube. 

Tube  No.  i.  0.5  c.c.  of  Immune  Serum  I,  diluted 
i -20. 

Tube  No.  2.    0.5  c.c.  of  Immune  Serum  II,  undiluted. 

Tube  No.  3.  0.5  c.c.  of  Imnrune  Serum  II,  diluted 
i -20. 

Tube  No.  4.    0.5  c.c.  of  Immune  Serum  III,  diluted 

1-5- 

Tube  No.  5.    o.i  c.c.  of  sterile  ox  bile. 

Prepare  the  bile  by  autoclaving,  filter  the  precipitate, 
and  again  autoclaving. 

Incubate  tubes  one  hour  at  37°  C.,  then  make  readings. 

Types  I,  II,  or  III  are  identified  by  the  appearance  of 
clumping  or  agglutination  of  the  organisms.  If  there 
is  no  agglutination  in  the  tubes  containing  the  immune 


Sputum  89 

serum  and  the  bacteria  in  the  bile  tube  are  found  upon 
microscopical  examination  to  be  dissolved,  then  the  or- 
ganism is  identified  as  Type  IV.  All  strains  of  pneti- 
mococcus  are  dissolved  by  bile,  while  all  strains  of  strep- 
tococcus are  not;  therefore  Tube  No.  5  prevents  the 
mistake  of  classifying  a  streptococcus  as  Type  IV 
pneumococcus. 

Make  cultures  from  the  heart  blood  of  the  mouse  on 
blood  agar  plate  and  in  bouillon  for  control.  With  a  pure 
bouillon  culture  carry  out  the  same  procedure  as  in  the 
bacterial  suspension.  Follow  the  same  procedure  with 
10  c.c.  of  the  patient's  blood,  if  a  positive  culture  has 
been  obtained,  also  positive  cultures  of  pneumococci 
from  spinal  fluids  and  other  material. 

The  culture  media  used  should  be  made  from  meat 
infusion  0.3  to  0.5  acid  to  phenolphthalein,  sterilized  in 
an  Arnold  sterilizer  for  twenty  minutes  on  three  succes- 
sive days.  The  addition  of  5  c.c.  of  sterile  defibrinated 
rabbit  blood  to  the  bouillon  and  agar  favors  the  growth 
of  the  pneumococcus. 

Sputum 
Name Date 

Macroscopic  Examination:  Microscopic  Examination: 

Color  Blood  Cells 

Odor  Pus  Cells 

Consistence  Casts 

Blood  Elastic  Tissue 

Character —  Parasites 

Mucous  Tubercle  Bacilli 

Muco-purulent  Other  Bacteria 

Purulent 

Serous 

REMARKS  . 

Signed 


CHAPTER  VII 
THE  BLOOD 

The  Blood  is  an  opaque,  viscous  fluid,  and  the  viscosity 
or  measure  of  stickiness  of  the  blood  as  it  comes  in  con- 
tact with  the  vessel  walls  is  increased  by  a  meat  diet  and 
decreased  by  the  abundant  use  of  fluids.  It  consists  of  a 
colorless  liquid,  the  plasma,  containing  erythrocytes,  leu- 
cocytes, blood-plates,  and  blood-dust 

Color 

Dark  red,  due  to  the  presence  of  red  blood  corpuscles. 
The  redder  it  is,  the  richer  it  is  in  oxyhemoglobin ;  the 
darker,  the  greater  amount  of  reduced  hemoglobin. 

Pathogenic  Changes  in  Color 

Cherry  red  in  coal-gas  poisoning. 

Brownish  red  or  chocolate  color  in  poisoning  from 
aniline,  potassium  chlorate,  hydrocyanic  acid,  and  nitro- 
benzol. 

Odor 

The  odor  is  characteristic  and  is  due  to  volatile  and 
fatty  acids. 

Taste 

Salty  and  at  the  same  time  insipid. 

90 


The  Blood  QI 

Specific  Gravity 

This  varies  with  the  amount  of  hemoglobin  and  is  in- 
fluenced by  age  and  sex,  digestion,  exercise,  pregnancy, 
etc. 

Average  in  adults  is  1.058  to  1.062. 

Amount 

The  total  amount  of  blood  in  the  normal  adult  is  said 
to  amount  to  ^V  of  the  body  weight. 

Reaction 

Alkaline,  due  to  mono-sodium  carbonate  and  di-sodium 
phosphate  in  solution  in  the  blood. 

Laked  Blood 

Laked  blood  or  hemolysis  is  the  dissolution  of  the 
blood  corpuscles.  The  red  blood  cells  are  separated  from 
the  hemoglobin  and  rendered  transparent  by  the  action 
of  certain  substances,  such  as  chloroform,  ether,  or 
water. 

The  use  of  a  0.3%  acetic  acid  solution  as  a  diluting 
fluid  for  the  purpose  of  counting  the  white  corpuscles 
lakes  the  red  corpuscles,  and  it  not  only  preserves  the 
white  cells,  but  it  also  makes  their  nuclei  more  distinct. 

Erythrocytes 

The  number  of  erythrocytes  or  red  blood  corpuscles 
present  in  the  fluid  obtained  from  well-developed  males 
in  good  physical  condition  is  5,500,000  per  cubic  milli- 
meter, and  the  normal  content  of  the  blood  of  females  is 
from  4,000,000  to  4,500,000  per  cubic  millimeter. 

Their  source  is  in  the  bone  marrow.  They  are  ^Aff 
of  an  inch  in  diameter.  Microscopically  they  appear  as 
non-nucleated  biconcave  discs  of  a  pale  hue. 


92  Clinical  Laboratory  Technic 

Function 

1.  To  carry  oxygen  from  the  lungs  to  the  tissues.    - 

2.  To  carry  carbon  dioxide   from  the  tissues  to  the 
lungs. 

The  erythrocytes  may  be  increased  after  transfusion, 
by  residing  in  a  high  altitude,  as  a  result  of  strenuous 
exercise,  in  starvation,  after  meals,  after  hot  and  cold 
baths,  after  the  administration  of  certain  drugs  and 
radium,  and  accompanying  certain  diseases,  such  as 
cholera,  diarrhea,  yellow  atrophy  of  the  liver. 

A  decrease  occurs  in  the  different  forms  of  anoemia 
(  oligocythemia) . 

Polycythemia — an  increase  in  the  number  of  red  blood 
cells. 

Anisocytosis 

A  term  used  where  there  is  marked  variation  in  the 
size  of  the  erythrocytes.  Macrocytes  are  large  red  cells 
and  are  found  in  severe  forms  of  anemia.  Microcytes  are 
small  red  cells  found  in  the  less  grave  types  of  anemia. 
Poikilocytes  are  red  cells  which  have  become  distorted 
in  shape.  Pencil  forms  are  slender,  elongated  red  cells,, 
characteristic  of  chronic  secondary  anemia. 

Polychromatophilia 

Red  blood  cells  which  show  polychromatophilia  or 
various  tints,  brownish  to  a  bluish-gray  tint,  occur  in 
malaria,  leukemia,  and  pernicious  anemia. 

Achromia 

Red  blood  cells  showing  achromia  are  characterized 
by  pallor  of  the  center  of  the  stained  cells.  These  cells 
are  common  in  anemias. 


The  Blood  93 

Stippled  Cells 

Stippling  or  granular  basophilic  degeneration  is  the 
presence  of  irregular  basophilic  granules  in  the  stained 
red  blood  cell.  These  cells  occur  in  chronic  lead  poison- 
ing, severe  anemias,  as  pernicious  anemia,  malarial 
cachexia,  and  the  leukemias. 

Howell- Jolly  Bodies 

Small,  round  nuclear  particles  which  stain  a  deep 
purple  with  Wright's  stain.  They  are  found  in  perni- 
cious anemia.  The  greatest  numbers  are  seen  after 
splenectomy. 

Cabot's  Rings 

These  are  loops  or  figures  of  eight  shaped  structures 
which  are  seen  in  stained  red  blood  cells  in  lead  poison- 
ing, leukemia,  and  pernicious  anemia. 


0  # 


ERYTHROCYTES  — After  Gibson 

I,  Normal  cells;  2,achromia;  3, stippling;  4,  microcyte  ;  5,macrocyte  ; 
6,  poikilocytes  ;  7,  pencil  forms  ;  8,  normoblasts  ;  9,  megaloblasts  ; 
10,  Cabot's  rings  ;  1 1,  Howell- Jolly  bodies ;  12,  reticulated  cells. 

Erythroblasts 

Normoblasts  are  about  the  size  of  a  normal  red 
blood  cell,  with  a  round  nucleus  which  stains  intensely. 
They  are  found  in  myelogenous  leukemia  and  secondary 
anemias. 


94  Clinical  Laboratory  Technic 

Megaloblasts  are  larger  than  the  normal  red  cell,  and 
the  nucleus  is  often  irregular  and  stains  less  intensely. 
In  myelogenous  leukemia  these  cells  are  often  seen  with 
a  bluish-gray  cytoplasm.  They  are  characteristic  of 
pernicious  anemia.  Gigantoblast  is  a  name  given  to  large 
megaloblasts. 

Hematoblasts 

These  cells  are  sometimes  called  proerythroblasts. 
They  resemble  lymphocytes.  Some  have  a  definite  ne- 
cleolus  and  a  bluish-gray  protoplasm. 

Leucocytes 

The  leucocytes,  or  white  blood  corpuscles,  differ  from 
the  erythrocytes  in  being  larger  and  containing  a  nucleus. 
The  normal  number  varies  between  5,000  and  10,000  per 
cubic  millimeter.  Microscopically  they  appear  as  white, 
spheric,  ameboidal  masses  of  protoplasm. 

Function 

1.  To  protect  the  body  against  pathogenic  bacteria. 

2.  To  aid  in  the  absorption  of  the  fats  and  peptones 
from  the  intestines. 

3.  To  take  part  in  the  process  of  blood  coagulation. 

4.  To  help  maintain  the  normal  composition  of  the 
blood  plasma  as  to  proteins. 

A  leucocytosis  is  an  increase  in  leucocytes.  Physiolog- 
ical leucocytoses  accompany  pregnancy,  parturition,  and 
digestion,  as  well  as  those  due  to  thermal  influences. 

Pathological  leucocytoses  occur  in  inflammatory,  in- 
fectious, post-hemorrhagic,  and  toxic  conditions. 

Leucopenia 

A  decrease  in  leucocytes,  as  in  typhoid  fever. 


The  Blood  95 

Blood  Plasma,  the  intercellular  substance  of  the  blood. 

Blood  Serum  is  the  light  yellow  fluid  which  exudes 
after  the  blood  coagulates  and  the  clot  forms.  It  is  the 
plasma  deprived  of  its  fibrin. 

Blood-dust 

Hemokonia,  or  blood-dust,  is  one  of  the  form  elements 
held  in  suspension  in  the  liquid  called  blood  plasma,  and 
appears  as  small  hyaline,  refractive  bodies. 

The  Opsonic  Index 

Opsonin  is  the  substance  developed  in  the  blood  when 
the  body  is  invaded  by  bacteria,  and  this  substance  in- 
creases the  phagocytic  activity  of  the  leucocytes  for  this 
particular  organism. 

The  opsonic  index  is  the  measure  of  this  activity. 

Bacteriology  of  the  Blood 

Pyogenic  and  other  organisms  are  often  found  by 
direct  inoculation  of  ordinary  culture  media  with  blood. 

Blood  cultures  are  valuable  in  the  following  diseases : 
typhoid  fever,  pneumonia,  pelvic  diseases,  endocarditis, 
local  diseases  of  the  throat,  and  malaria. 

A  vein  in  the  arm  is  punctured  under  strict  aseptic 
precautions.  Scrub  the  arm  2  inches  above  and  2  inches 
below  the  elbow  with  soap  and  water,  then  alcohol.  In- 
sert a  sterile  needle  in  median  or  basilic  vein.  Use  sterile 
test  tube  for  receptacle. 

Plant  in  fluid  media  or  agar  plates,  or  take  2^  c.c.  of 
blood  and  add  to  5  c.c.  of  sterile  ox  bile,  and  place  this 
in  a  thermostat  at  120°  F.  Then  take  2  loopfuls  of  the 
blood  ox  bile  and  inoculate  a  tube  of  bouillon,  and  incu- 
bate over  night. 


96  Clinical  Laboratory  Technic 

If  a  motile  bacillus  develops,  it  is  probably  typhoid, 
and  can  be  determined  by  a  Widal  test. 

Coagulation 

Normal  coagulation  occurs  in  about  3  to  5  minutes. 
The  clotting  of  blood  depends  upon  the  conversion  of 
fibrinogen  into  fibrin. 

Method  of  Determining  the  Rate  of  Coagulation 

Puncture  the  lobe  of  the  ear,  then  take  a  piece  of 
capillary  glass  tubing  and  holding  it  downward  from  the 
puncture  let  it  fill  for  3  or  4  inches.  At  intervals  of 
30  seconds  scratch  the  capillary  tubing  at  short  distances 
and  break  off  between  the  fingers. 

When  coagulation  has  taken  place,  a  long,  worm-like 
coagulum  is  formed. 

Color  Index 

In  normal  blood  this  is  approximately  i.  To  obtain  the 
color  index  divide  the  percentage  of  the  hemoglobin  by 
the  percentage  of  red  blood  cells,  5,000,000  red  cells  being 
considered  as  100%.  To  obtain  the  percentage  of  red 
cells,  multiply  the  two  extreme  figures  to  the  left  by  2. 
EXAMPLE.  A  count  shows  the  presence  of  2,400,000  red 
cells;  the  percentage  would  be  48  (24  X  2)«  The  hemo- 
globin percentage  is  72 ;  then  the  color  index  would  be 
72  divided  by  48,  or  1.5. 

Sahli's  Hemoglobinometer  for  Obtaining  the  Percent- 
age of  Hemoglobin  in  the  Blood. — Method 
First  wash  off  the  lobe  of  the  ear  with  alcohol,  then 
take  hold  of  the  lower  end  with  the  thumb,  first  and  second 
fingers.  x  Insert  a  glover's  needle  quickly.    Wipe  off  the 
first  drop  of  blood.    The  second  drop  is  drawn  up  by 
suction  into  the  pipette  to  the  20  cmm.  mark. 


The  Blood 


97 


Pour  N/io  HC1  into  the  graduated  tube  to  the  mark  10 
on  the  scale  of  the  tube,  then  add  to  this  the  20  cmm.  in 
the  pipette.  When  the  mixture  assumes  a  clear,  bright 
color,  add  distilled  water,  drop  by  drop,  until  the  color 
matches  the  color  in  the  standard  tube. 

Talquist's  Hemoglobin  Scale 

This  is  a  book  of  specially  prepared  filter  paper,  with 
a  scale  of  ten  shades  of  blood  colors.  These  are  so  tinted 
as  to  match  blood  taken  up  on  a  piece  of  filter  paper,  and 
are  graded  from  10  to  100.  The  comparison  should  be 
made  as  soon  as  the  blood  on  the  filter  paper  has  lost  its 
humid  gloss. 

Oligochromemia 

A  diminution  in  the  normal  amount  of  hemoglobin. 

Hemacytometers  or  Blood  Counters 

These  instruments  are  used  for  determining  the  numer- 
ical ratio  between  the  red  and  white  corpuscles  contained 


H  EM  ACYTOMETERS 


in  the  human  blood.  Undiluted  blood,  owing  to  the  ex- 
cessively large  numbers  of  blood  discs  contained  in  a 
single  cubic  millimeter,  is  unsuitable  for  counting,  and 
it  is  necessary  to  dilute  the  blood  in  certain  proportions. 


98  Clinical  Laboratory  Technic 

A  definitely  determinable  dilution  is  obtained  with  the  aid 
of  the  mixing  pipettes. 

The  melanger  or  large  pipette  is-  used  for  counting  the 
red  corpuscles,  and  gives  a  dilution  of  i :  200  when  the 
blood  is  drawn  up  to  the  mark  0.5. 

Method 

Cleanse  the  lobe  of  the  ear  with  alcohol  and  insert  a 
sterile  glover's  needle  or  a  straight  Hagedorn  needle  by 
a  rapid  puncture.  The  first  drop  of  blood  should  be  wiped 
away,  and  as  soon  as  the  blood  is  flowing  freely,  put  the 
point  of  the  pipette  into  the  drop  as  it  emerges  from  the 
ear ;  by  sucking  gently  on  the  rubber  attached  to  the  other 
end,  draw  up  the  blood  to  the  mark  0.5  on  the  pipette. 
Wipe  the  end  of  the  pipette,  then  plunge  the  point  into 
Gower's  solution  and  fill  the  bulb  up  to  the  mark  101. 
Thoroughly  mix  the  blood  by  rolling  the  pipette  between 
the  palms. 

The  small  pipette  is  used  for  counting  the  white  cor- 
puscles and  gives  a  dilution  of  i :  20  when  the  blood  is 
drawn  up  to  the  mark  0.5.  The  bore  of  this  tube  being 
large,  it  fills  and  empties  more  readily,  therefore  it 
should  be  kept  in  a  horizontal  position.  Draw  the  blood 
up  to  the  mark  0.5  on  the  pipette,  wipe  the  end  of  the 
pipette,  then  plunge  the  point  into  0.3%  acetic  acid,  and 
fill  the  bulb  up  to  the  mark  n. 

When  the  white  count  is  very  high,  the  red  counter 
may  be  used,  diluting  the  blood  200  times  instead  of  20. 
This  must  be  remembered  in  determining  the  final  count. 

Clean  the  pipettes  with  water,  then  alcohol  and  ether. 
The  mouthpieces  attached  to  the  rubber  tubing  should  be 
boiled  often  and  kept  in  95%  alcohol. 


The  Blood  99 

The  Counting  Chamber 

The  number  of  corpuscles  contained  in  a  cubic  milli- 
meter of  the  dilute  solution  of  blood  is  determined  by 
counting  in  a  cell  of  known  depth  and  area.  The  count- 
ing chamber  consists  of  a  plane  glass  plate  with  a  circular 
hole,  which  being  cemented  to  a  plane  slide  of  stout  glass 
forms  a  well.  The  bottom  of  the  latter  is  formed  by  a 
disc,  the  thickness  of  which  is  exactly  o.i  mm.  less  than 
that  of  the  perforated  glass. 

The  counting  chamber  consists  of  16  squares  of  J  mm. 
side,  each  of  which  is  again  divided  into  25  small  squares 
having  sides  -£$  mm.  The  area  of  each  of  the  small 
squares  is  -^fa  square  millimeter,  and  the  distance  between 
the  floor  and  the  lower  surface  of  the  cover  is  ^  mm.; 
therefore,  each  square  represents  3^0 tf  cubic  millimeter. 

To  clean  the  counting  chamber,  rinse  in  cold  water  and 
dry  carefully. 

Adjusting  a  Drop  of  Blood 

1.  Place  the  cover  glass  (an  optically  plane  glass)  in 
position;  then  by  slightly  pressing  with  the  rubber  end 
of  a  pencil,  concentric  rainbow  rings  will  be  seen.    These 
are  known  as   Newton's  rings,  and  must  remain  after 
pressure  is  removed,  otherwise  there  is  dust  under  the 
glass. 

2.  Blow  out  3   drops  of  the  diluted  solution  in  the 
pipette. 

3.  Place   a   drop   upon  the   surface  of  the   counting 
chamber  of  such  a  size  that  when  the  cover  glass  is  let 
down  over  it  the  whole  of  the  disc  is  covered  without  any 
being  spilled  into  the  moat  around  it. 

4.  Place  the  cover  glass  over  the  drop  before  the  cor- 
puscles have  time  to  settle. 


IOO 


Clinical  Laboratory  Tcchnic 


5.    After  waiting  a  minute  for  the  corpuscles  to  settle, 
if  the  distribution  seems  uniform  the  counting  is  begun. 

Register  for  Recording  the  Number  of  Blood  Cells 
Counted 

Place  the  index  finger  of  the  left  hand  through  the  ring, 
holding  the  Register  in  position  so  as  to  bring  the  pusher 
directly  under  the  thumb.  In  commencing  to  count,  only 
the  o's  should  appear ;  thus,  ooo.  The  Register  will  count 
to  999 ;  the  next  count  causes  the  ooo's  to  appear,  making 
i, ooo.  To  set  the  Register  at  o,  press  i-ie  pusher  down 
the  required  number  of  times  until  the  o  appears  at  the 
right,  and  then  change  the  other  figures  at  the  left  by 
turning  the  thumb  buttons  on  back  of  the  Register  till  the 
o's  appear,  always  turning  the  thumb  button  of  middle 
dial  first. 
Counting  the  White  Blood  Corpuscles 

Count  all  the  corpuscles  in  the  400  small  squares,  ruling 


out  those  touching  the  outside  lines.  Count  four  separate 
drops,  then  divide  by  four  to  get  an  average;  multiply 
the  result  by  the  dilution  (  ^ » then  by  4,000  (each  square 
is  equivalent  to  ¥oVo  of  a  cubic  millimeter),  and  divide 


The  Blood  101 

by  400  (the  number  of  squares  counted).     The  result 
will  be  the  number  of  corpuscles  per  cubic  millimeter. 

EXAMPLE.      Average   number   of   corpuscles   in   four 
drops  is  45. 


45  X  20  X  4,000 

=  0,000 

400 


Counting  the  Red  Corpuscles  ••••••• 

Count  all  the  corpuscles  in  the  25  small  squares,  begin- 
ning at  the  upper  left-hand  corner  (a),  counting  the  25 
small  squares,  then  the  upper  right-hand  corner  (&),  the 


lower  right-hand  corner  (c),  and  the  lower  left-hand 
corner  (d),  making  a  total  count  of  100  small  squares. 

The  sum  of  all  the  corpuscles  in  the  100  small  squares 
is  multiplied  by  the  dilution  Gtro)>  then  by  4,000,  and 
divided  by  100  (the  number  of  squares  counted). 

EXAMPLE.  The  number  of  corpuscles  counted  in  100 
squares  is  685. 

685  X  200  X  4,000 

-  =  5,480,000 


IO2  Clinical  Laboratory  Technic 

Vital  Staining  of  Blood.      (Journal  A.  M.  A.,  March, 


Stain  fresh  without  fixation.     The  stains  may  be  dis- 

solved in  physiological  salt  solution,  Ringer-Locke  solu- 

tion, or  even  directly  in  the  blood  itself.    The  blood  is 

mixed  with  the  stain  and  examined  immediately,  prefer- 

^jably  on.  aj  \Va;rin  ;stage. 

„  ..The^staining1  solution  may  be  placed  on  the  glass,  the 
rnai  gin  ;Hqing  .sealed  with  paraffin  or  balsam,  to  prevent 
evaporation. 

In  typical  vital  staining  the  nucleus  is  not  stained,  only 
certain  granules  and  fibers  in  the  cytoplasm  taking  the 
stain.  As  the  cell  begins  to  die,  "post-vital  staining  of 
whole  or  parts  of  the  cells  may  occur.  Lymphocytes  are 
recognized  by  the  size  and  shape  of  the  nuclei  ;  neutrophile 
leucocytes,  by  their  fine  granulations;  eosinophiles,  by 
their  coarser  granules. 

Among  the  stains  used  in  vital  staining  are  methyl 
violet,  neutral  red  fuchsin,  toluidine  blue,  thionine,  and 
Nile  blue;  o.i  c.c.  of  any  one  of  these  stains  to  250  c.c. 
of  water  or  salt  solution. 

In  studying  mitochondria,  or  small,  rod-shaped  gran- 
ules in  the  cytoplasm  of  leucocytes,  a  ^-$1^  solution  of 
Janus  green  is  used.  The  Janus  green  (diethylsafrani- 
nazo-dimethylanilin)  is  dissolved  in  0.85%  salt  solution. 

Modification  of  Ringer-Locke  Solution 

Sodium  chloride  6.    c.c. 

Calcium  chloride  0.2  c.c. 

Potassium  chloride  0.4  c.c. 

Sodium  carbonate  0.2  c.c. 

Grape  sugar  i.oc.c. 

Distilled  water  1,000.    c.c. 


The  Blood  103 

Staining  Reticulated  Cells 

Many  of  these  cells  are  present  in  anemic  blood. 

METHOD.  Place  one  small  drop  of  i%  aqueous  solu- 
tion of  brilliant  cresyl-blue  upon  a  slide.  Allow  to  dry 
in  such  a  manner  that  a  thin  film  of  the  dye  is  obtained. 
A  small  drop  of  blood  on  a  cover-glass  is  placed  upon 
the  dried  dye.  Allow  the  blood  to  spread,  using  gentle 
pressure  if  necessary. 

Examine  with  the  oil  immersion  objective.  A  blue 
staining  reticulum  of  delicate,  contorted  filaments  will 
be  seen  within  the  red  blood  cell:  Platelets  appear  as 
irregular  shaped  hyaline  bodies,  with  a  round,  blue- 
staining,  granular  central  structure.  The  white  blood 
cells  stain  blue. 

Mechanical  Stage 

This  stage  is  a  great  aid  to  exact  work  in  differential 
counting.  It  is  attached  to  the  stage  of  the  microscope 
by  two  set  screws.  By  rack  and  pinion  giving  equal  speed 
in  both  movements,  right  and  left  scale  reads  60  mm.; 
backward  and  forward,  no  mm.  It  is  provided  with  one 
adjustable  and  one  fixed  stop,  which  is  actuated  by  a 
spring  to  hold  the  slide  in  place. 

Smears  for  Differential  Count 

Cleanse  the  ear  lobe  with  alcohol,  and  after  puncturing 
it  with  a  sterile  needle  wipe  off  the  first  drop.  A  clean 
slide  is  held  without  touching  its  surface,  and  touched  to 
the  summit  of  the  drop  as  soon  as  it  emerges.  This  drop 
is  then  drawn  out  smoothly  over  the  glass  with  the  edge 
of  another  slide. 

The  slide  should  not  come  in  contact  with  the  skin 
while  it  is  being  charged  with  the  blood,  and  no  pressure 
should  be  used  in  spreading  the  drop. 


IO4  Clinical  Laboratory  Technic 

Dry  quickly  by  waving  the  slide  in  the  air. 

Cover  the  smear  with  Wright's  stain  for  i  minute. 

Add  same  amount  of  water  to  the  stain,  let  this  remain 
3  minutes,  then  wash  the  slide  with  water. 

A  differential  count  of  400  cells  should  be  counted  to 
secure  reliable  results.  The  percentage  value  of  each 
variety  is  then  figured. 

Normal  varieties  and  figures  in  I  cmm.  of  blood : 

Polymorphonuclear  neutrophiles  65-70% 

Lymphocytes  20-35% 

Transitionals — large  mononuclears  3~  5% 

Eosinophiles  2-  4% 

Mast  cells  \% 

While  making  the  differential  count,  the  variations  in 
size  of  the  red  blood  cells,  achromia,  and  polychromato- 
philia,  and  the  number  of  blood  platelets  should  be  noted. 

Cover-glass  Method  for  Smears 

Touch  the  center  of  the  cover-glass  to  drop  of  blood 
from  the  ear,  then  drop  immediately  on  another  cover- 
glass  and  allow  to  spread.  Separate  by  sliding  one  cover- 
glass  from  the  other. 

Erythrocytes.— Wright's  Stain 

a.  Normal  red  blood  cells. 

b.  Microcytes,  small  red  blood  corpuscles. 

c.  Crenated  cells.     If  the  density  of  the  plasma  is  in- 
creased in  any  way,  as  by  evaporation,  many  of  the  red 
cells  become  shrunken  and  crenated  by  the  passage  of 
water  out  of  the  corpuscle. 

d.  Macrocytes,  large  red  blood  corpuscles. 


e 


n 


a 


n 


c 


b 


'•*> 


5B»     ^  ^I^Sf.     <W 

e  f 


* 


The  Blood  105 

e.  Poikilocytes,  malformed,  pear-shaped  red  cells. 

/.  Red  cells  showing  degenerative  changes. 

g.  Normoblast,  a  nucleated  red  cell  of  the  ordinary 
size. 

h.  Megaloblast,  a  large  nucleated  red  cell. 

i.  Amitosis,  or  direct  cell  division. 

/.  Free  nucleus. 

k.  Polychromatophilia,  various  stains  or  tints. 

/.  Achromia,  absence  of  color. 

m.    Stippled  cells,  characteristic  of  lead  poisoning. 

n.  Blood  platelets. 

o.  Karyokinesis,  mitotic  or  indirect  cell  division. 

p.  Malarial  organisms, 

Leucocytes. — Wright's  Stain 

a.  Lymphocytes. 

b.  Large  mononuclears. 

c.  Polymorphonuclear  neutrophiles. 

d.  Eosinophiles. 

e.  Mast  cells. 

/.  Myelocytes,  neutrophilic. 
g.  eosinophilic. 

h.  basophilic. 

NOTE. — Neutrophilic  granules  are  purple ;  basophilic,  dark  blue ; 
acidophilic,  red. 

Leucocytes. — Wright's  Stain 

Lymphocytes 

These  cells  are  about  the  size  of  a  red  blood  cell,  and 
are  derived  from  the  lymph  glands.  They  have  a  large 
nucleus,  with  small  margin  of  protoplasm.  The  nucleus 
stains  intensely,  while  the  protoplasm  is  light  blue  and 
is  usually  free  from  granules. 


io6  Clinical  Laboratory  Technic 

Large  Mononuclears 

Normally  these  cells  have  a  round  or  oval  nuclei  which 
does  not  stain  so  deeply  as  the  lymphocytes,  and  they 
possess  more  protoplasm.  At  a  later  stage  in  the  decay 
of  these  cells,  the  nuclei  are  more  indented,  frequently 
horseshoe-shaped,  stain  less  intensely,  and  have  a  very 
granular  protoplasm. 

Polymorphonuclear  Neutrophiles 

These  cells  have  a  multiple,  irregular-shaped  nucleus. 
The  nucleus  stains  deeply  and  the  protoplasm  is  dotted 
with  neutrophilic  granules.  They  are  derived  from  the 
neutrophilic  myelocytes  of  the  bone  marrow. 

Eosinophiles 

These  cells  have  an  irregular-shaped  nucleus,  and  the 
protoplasm  is  covered  with  coarse  acidophilic  granules 
which  are  highly  refractive.  They  are  formed  from  the 
acidophilic  myelocytes  of  the  bone  marrow. 

Mast  Cell 

The  nucleus  of  this  cell  is  multiple  and  irregular,  and 
the  protoplasm  is  covered  with  large  basophilic  granules. 
They  are  formed  from  the  basophilic  myelocytes. 

Pathogenic  Leucocytes 

Myelocytes 

NEUTROPHILIC. — The  nucleus  is  large  and  irregular, 
and  the  protoplasm  is  covered  with  neutrophilic  granules. 

EOSINOPHILIC. — This  cell  has  a  large  oval  nucleus, 
and  the  protoplasm  is  covered  with  eosinophilic  granules. 

BASOPHILIC. — The  nucleus  of  this  cell  is  large  and  the 
protoplasm  is  covered  with  small  basophilic  granules. 


The  Blood  107 

Myeloblasts 

These  cells  are  often  mistaken  for  large  lymphocytes. 
They  are  the  lymphoid  cells  of  the  bone  marrow  and  are 
the  parent  cells  of  the  myelocytes.  The  nucleus  stains 
more  deeply  than  that  of  the  large  mononuclears,  and 
the  small  amount  of  protoplasm  is  stained  more  deeply 
blue.  There  are  no  granules.  They  also  show  three  or 
four  nucleoli  in  the  nucleus. 


•  •  * 


16        17 


19  •  EO 


LEUCOCYTES  —  After  Gibson 

13,  polymorphonuclear  neutrophiles  (abnormally  granular) ;  14, 
lymphocytes;  15,  mononuclears;  16,  myeloblast ;  17,  young  myelocyte; 
18,  megacaryocyte  imbedded  in  platelets;  19,  megacaryocyte-dividing 
nucleus  ;  20,  megacaryocyte  sending  out  pseudopodium. 

Megacaryocyte 

The  giant  cell  of  the  bone  marrow.  These  cells  are 
frequently  found  in  myelogenous  leukemia.  The  nucleus 
has  a  gnarled  appearance.  There  is  considerable  bluish 
protoplasm.  These  cells  are  often  seen  imbedded  in 
platelets. 

Blood  Diseases 
Anemia 

A  condition  in  which  the  quality  and  quantity  of  the 
blood  is  deficient. 


io8  ,    Clinical  Laboratory  Technic 

Pernicious  Anemia 

Leukopenia.  Color  index  is  high.  Red  blood  cells, 
2,000,000  to  200,000.  Marked  anisocytosis.  Megalo- 
blasts  increased.  Decrease  in  blood  platelets.  Poikilo- 
cytosis,  polychromatophilia,  and  stippling,  normoblasts, 
myelocytes,  and  myeloblasts  sometimes  present. 

Chlorosis.  Leucocytes  normal.  Color  index  is  low. 
Slight  anisocytosis.  Normoblasts. 

Splenomyelogenous  Leukemia  (Myeloid  Leukemia) 

The  red  blood  cell  count  is  about  3,000,000  and  the 
color  index  low.  The  leucocyte  count  is  on  the  average 
from  200,000  to  500,000,  may  go  as  high  as  1,000,000. 

Some  types  of  myelogenous  leukemia  have  a  low  white 
count,  although  the  spleen  and  liver  are  markedly  en- 
larged. Such  cases  show  a  large  number  of  cells  of 
mononuclear  type,  but  of  bone  marrow  origin  (abortive 
myelocyte). 

In  acute  cases  and  in  exacerbations  of  the  disease 
there  are  many  myeloblasts. 

Lymphatic  Leukemia.  Red  blood  count  about 
3,500,000.  Color  index  slightly  below  normal.  Small 
lymphocytes,  70-98%.  White  blood  count,  8,000  to 
250,000. 

In  acute  lymphatic  leukemia  the  large  lymphocyte 
predominates. 

Pseudoleukemias 

HODGKINS  DISEASE. — Increase  in  lymphocytes  in  early 
stage  with  few  abnormal  mononuclears.  Mononuclears 
are  generally  very  granular,  and  increase  as  the  disease 
progresses.  Eosinophiles  increased.  Blood  platelets  are 
increased.  Marked  decrease  in  last  stages. 


The  Blood  109 

LYMPHOSARCOMA. — Marked  anemia.  Neutrophilic 
leucocytosis.  Decrease  in  lymphocytes. 

LYMPHOID  PSEUDOLEUKEMIA. — 5,000  to  14,000  white 
blood  count,  with  70%  lymphocytes. 

Multiple  Myeloma 

Increase  of  Tiirck's  irritation  forms.  (Test  urine  for 
Bence-Jones  body.) 

Granulomatoses 

Neutrophilic  leukocytosis. 

Oxydase  Reaction.     Graham's  Stain 

METHOD. — Fresh  smears  are  preferable.  Dry  thor- 
oughly in  the  air. 

1.  Fix  smears   for  2  minutes  in  a   freshly  prepared 
mixture  of  9  parts  of  95%  alcohol  and  I  part  of  strong 
formaldehyde  solution. 

2.  Wash  in  water. 

3.  Flood  with  the  following  alphanaphthol  solution: 

Alphanaphthol  (Merck's  Reagent)  I  gm. 

Alcohol  (40%)  i  oo  c.c. 

Hydrogen  peroxide  0.2  c.c. 

4.  Allow  a  reaction  time  of  5  minutes. 

5.  Wash    and    place   in    dish   of    running   water    15 
minutes. 

6.  Stain  with  the  following  solution: 

Pyronin  o.i  gm. 

Alcohol  (40%)  96  c.c. 

Aniline  4  c.c. 

Dissolve  the  pyronin  in  the  alcohol,  then  add  the 
aniline. 

7.  Wash  in  water. 


no  Clinical  Laboratory  Technic 

8.  Stain  I  minute  with  a  0.5%  aqueous  solution  of 
methylene-blue  (Griibler's  B.  X.). 

9.  Wash  in  water,  blot,  and  dry. 

10.  Mount  in  neutral  balsam. 

The  neutrophilic  granules  stain  a  purplish-red  in  color, 
and  are  usually  very  abundant.  Old  and  degenerating 
forms  show  fewer  granules.  The  mast  cells  take  a  more 
basic  stain.  The  eosinophilic  granules  are  larger  and 
lighter  and  more  refractile,  with  lighter  staining  centers. 
The  granules  in  the  myelocytes  vary  in  number  and  size. 
Nuclei  of  all  cells  appear  blue,  and  cytoplasm,  light  blue. 

Red  blood  cells  appear  greenish-yellow. 

Platelets  are  blue. 

This  stain  is  of  value  in  differentiating  myelogenous 
from  lymphatic  leukemia. 

Fragility  of  Red  Cells 

There  is  less  resistance  to  hypotonic  salt  solution  in 
cases  of  hemolytic  jaundice,  and  an  increased  resist- 
ance in  obstructive  jaundice  (cancer  of  pancreas  and 
gallstones).  Pernicious  anemia  shows  an  increased 
resistance. 

Fragility  Test 

Withdraw  6  c.c.  of  blood  from  a  vein  in  the  arm  and 
transfer  to  a  test  tube  half  full  of  0.5%  sodium  citrate 
solution  in  0.9%  sodium  chloride.  Mix  by  inverting 
tube  several  times.  Centrifuge,  pour  off  supernatant 
fluid,  and  wash  the  cells  twice  with  0.7%  sodium  chloride. 
Draw  off  as  much  supernatant  fluid  as  possible  with  a 
pipette  and  use  the  remaining  blood  cells  for  the  test. 

Make  hypotonic  sodium  chloride  solutions  from  a  i% 
solution  of  chemically  pure  sodium  chloride  and  distilled 


The  Blood  HI 

water.  The  solutions  are  kept  in  tightly  corked  100  c.c. 
bottles  and  run  in  strength  from  0.70  to  0.175%.  With 
a  pipette,  draw  off  i  c.c.  and  transfer  to  a  series  of  small 
test  tubes  (i  c.c.  for  each  tube),  then  add  0.05  c.c.  of 
blood  cells  to  each  tube.  Invert  each  tube  twice  and 
allow  to  stand  two  hours  at  room  temperature.  Make  a 
reading  of  the  tubes.  The  point  at  which  there  is  the 
first  tinge  of  pink  in  the  salt  solution  is  considered  the 
initial  hemolysis,  and  the  point  at  which  there  can  no 
longer  be  seen  any  sediment  of  blood  is  the  complete 
hemolysis. 

The  average  points  of  hemolysis  as  obtained  by  this 
method  are  (from  Hill)  : 

Normal  blood  .457   (initial)   3.40     (complete) 

Secondary   anemia      .475   (initial)     .322   (complete) 
Pernicious  anemia       .477   (initial)     .322   (complete) 
Chronic  family 

(hemolytic)   jaundice  .600   (initial)     .400  (complete) 
Obstructive  jaundice  .400  (initial)      .225   (complete) 

Matching  Bloods  for  Transfusion. — Minot's  Modi- 
fication of  Moss'  Method 

1.  Red  Blood  Cell  Suspension. — Puncture  the  ear  of 
each  of  the  two  persons  whose  blood  is  to  be  matched 
and  let  a  large  drop  of  blood  fall  directly  into  a  small 
test  tube  containing  i  c.c.  of  a  1.5%  solution  of  sodium 
citrate  in  0.9%  salt  solution.     Mix  by  inverting  several 
times. 

2.  Serum. — A  few  drops  of  blood  are  collected  in  a 
small  test  tube  or  a  Wright's  capsule.    Allow  to  clot,  then 
loosen  clot  from  the  wall  of  the  tube.     Let  stand  until 
the  serum  has  separated  well. 


112  Clinical  Laboratory  Technic 

3.  Make  vaseline  rings  on  2  slides.  In  the  ring  mix 
one  drop  of  the  patient's  serum  and  one  drop  of  the 
donor's  red  blood  cell  suspension.  In  the  other  ring  mix 
one  drop  of  the  patient's  red  blood  cell  suspension  and 
one  drop  of  the  donor's  serum. 

Transfer  to  a  cover  glass,  invert  over  a  hanging-drop 
slide  so  that  the  drop  hangs  freely  within  the  hollow 
space.  Mix  the  corpuscles  at  intervals  by  tilting  the 
slide. 

Examine  with  the  low  objective  power. 

When  agglutination  occurs,  the  corpuscles  come  to- 
gether in  clumps  or  masses.  Clumping  occurs  within 
a  few  minutes,  but  it  is  safe  to  wait  one-half  hour.  In 
case  of  rouleau  remix  the  cells.  The  serum  of  the  patient 
must  not  agglutinate  the  corpuscles  of  the  donor. 

Hemolysis  Test 

It  is  advisable  to  do  a  Wassermann  test  on  all  donors. 
The  donor's  red  cells  should  be  tried  against  the  serum 
of  the  recipient,  and  the  patient's  cells  against  the  donor, 
to  prove  the  absence  of  hemolysing  or  agglutinating 
bodies. 

DONOR. — Withdraw  two  samples  of  5  c.c.  each  from 
the  arm  vein.  Place  one  in  a  test  tube  and  allow  to  clot. 
The  clot  is  loosened  from  the  walls  of  the  tube  and  the 
serum  separated  by  centrifugation.  Place  the  second 
sample  in  a  test  tube  half  full  of  .5  sodium  citrate  in 
90%  sodium  chloride.  Centrifuge,  then  pipette  off  the 
supernatant  fluid.  Wash  three  times  with  normal  salt 
solution. 

RECIPIENT. — Repeat  the  same  procedure  with  the  re- 
cipient's blood.  Place  variable  quantities  of  recipient's 


The  Blood  113 

serum  in  three  small  test  tubes.  Add  to  each  tube 
0.25  c.c.  of  a  2%  suspension  in  normal  salt  solution  of  the 
blood  cells  of  the  donor.  The  same  procedure  is  carried 
out  with  the  donor's  serum  and  the  blood  cells  of  the 
recipient. 

Controls  should  be  made  with  the  donor's  serum  and 
donor's  cells,  and  recipient's  serum  and  recipient's  cells. 
Controls  are  also  made  with  donor's  cells  in  normal  salt 
solution  and  recipient's  cells  in  normal  salt  solution. 
Add  normal  salt  solution  to  each  tube,  raising  the  total 
volume  to  0.5  c.c. 

Incubate  the  test  tubes  in  a  water  bath  for  2  hours. 
Make  readings.  The  controls  of  the  patient's  may  be 
omitted  if  the  amount  of  blood  taken  from  the  patient 
is  small.  It  is  advisable  to  place  the  tubes  in  the  ice  box 
over  night  and  make  a  second  reading  in  the  morning 
unless  the  case  is  urgent. 

Blood  Groups1 

GROUP  I  ($%  of  individuals).  Serum  agglutinates 
cells  of  no  other  group.  Cells  are  agglutinated  by  sera 
of  Groups  II,  III,  IV.  As  donors  may  be  used  for 
Group  I  only.  As  recipients  may  receive  from  Groups  I, 
II,  III,  IV. 

GROUP  II  (40%  of  individuals).  Serum  agglutinates 

cells  of  Groups   I,  III.     Cells  agglutinated  by  sera  of 

Groups  III,  IV.     As  donors  may  be  used   for  Groups 

I,  II.     As  recipients  may  receive  from  Groups  II,  IV. 

GROUP  III  (10%  of  individuals).    Serum  agglutinates 

1  Tests  have  shown  that  individuals  fall  into  4  groups. 


U4  Clinical  Laboratory  Technic 

cells  of  Groups  I,  II.  Cells  are  agglutinated  by  sera  of 
Groups  II,  IV.  As  donors  may  be  used  for  Groups  I,  III. 
As  recipients  may  receive  from  Groups  III,  IV. 

GROUP  IV  (45%  of  individuals).  Serum  agglutinates 
cells  of  Groups  I,  II,  III.  Cells  are  agglutinated  by  no 
group.  As  recipients  may  receive  from  Group  IV  only. 

It  is  preferable  to  have  donor  and  recipient  of  the 
same  group.  Two  known  sera,  Types  II  and  III,  re- 
spectively, are  sufficient  for  the  determination  of  the 
group  to  which  any  blood  cells  belong. 

Macroscopic  Blood  Test 

Place  a  drop  of  Serum  II  and  a  drop  of  Serum  III 
on  the  right  and  left  ends  of  a  slide,  respectively ;  add 
Yz  drop  of  blood  from  ear  to  each  drop  of  serum,  using 
clean  rod  each  time.  Mix  blood  and  serum.  If  agglu- 
tination occurs  there  will  be  a  granular  or  brick  dust 
appearance  of  the  drop  in  about  I  minute. 

Blood  Platelets 

These  are  circular  or  oval  discs  2-3  microns  in  diam- 
eter. They  are  punched-off  projections  of  giant  cells 
of  the  bone  marrow.  There  is  some  evidence  that  in 
shed  blood  they  take  part  in  the  process  of  coagulation. 
Normally  they  average  about  350,000  per  cubic  milli- 
meter. They  are  absent  in  purpura,  lymphatic  leukemia, 
and  acute  aplastic  anemia.  There  is  a  marked  increase 
in  myelogenous  leukemia  and  early  Hodgkins,  and  large, 
abnormally  granular  platelets  and  large  protoplasm  plate- 
let masses  are  often  seen. 


The  Blood 


Blood  Platelets. — Counting.     (Journal  A.  M.  A., 
May  20,  1911) 

The  blood  is  diluted  i :  100  in  the  pipette  used  for  count- 
ing red  blood  corpuscles.  The  diluting  solution  consists 
of  2  parts  of  an  aqueous  solution  of  brilliant  cresyl  blue 
(1:300)  and  3  parts  of  aqueous  solution  of  potassium 
cyanide  ( i :  1400),  and  these  solutions  are  kept  in  separate 
bottles  and  mixed  immediately  before  using.  The  cresyl 
blue  solution  should  be  kept  on  ice  and  the  cyanide  solu- 
tion should  be  made  up  every  ten  days ;  the  mixed  solution 
should  be  filtered  before  using. 

The  ordinary  blood-counting  chamber  is  used ;  count 
with  the  high  dry  objective.  After  the  chamber  is  filled, 
it  is  left  at  rest  for  10  minutes,  in  order  that  the  platelets 
may  settle. 

Agglutination  Test 

One  of  the  properties  of  the  blood  is  the  power  of 
agglutinating,  or  clumping  and  rendering  immobile  the 
bacteria  which  it  may  be 
called  upon  to  attack. 
This  property  also, 
though  present  at  all 
times,  is  capable  of  de- 
velopment and  increase, 
as  is  seen  in  the  course  WIDAL  REACTION 

of    various    diseases  ;    as,  A — B.  typhosus-  before  adding  ty- 

for    example,    in   typhoid      phoid  blood.     B-K  typical  reaction ; 
.,,         the  bacilli  collect  in  clumps,  1.  e.,  be- 

fever,  where  the  specific     come  u  agglutinated.» 
agglutinins    of    the    pa- 
tient's blood  are  so  developed  as  to  show  a  clumping  of 
typhoid  bacteria  when  the  patient's  serum  and  a  culture 
of  typhoid  bacteria  are  brought  together. 


Ii6  Clinical  Laboratory  Technic 

First  cleanse  the  lobe  of  the  patient's  ear  with  alcohol, 
then  make  a  small  incision  with  a  needle  or  stilette,  letting 
the  blood  flow  into  a  capillary  pipette.  When  this  clots,  the 
serum  is  blown  out  on  the  loop  of  a  sterile  platinum  wire 
and  added  to  the  fluid  culture.  To  obtain  a  fluid  culture 
of  typhoid  bacilli,  make  a  transplant  from  a  culture  on 
agar-agar  to  bouillon,  and  place  in  the  incubator  over 
night. 

Paratyphoid  Bacilli 

These  bacilli  differ  from  typhoid  bacilli  by  producing 
gas  in  glucose  media  and  by  showing  different  agglutina- 
tion reactions.  The  two  types  generally  recognized  are 
known  as  "A"  and  "B." 

Type  "A,"  in  all  other  respects,  is  like  the  typhoid 
bacillus. 

Type  "B"  makes  milk  alkaline,  but  does  not  coagulate 
it,  and  after  eight  or  ten  days  it  becomes  translucent. 

Bacillus  Coli  Communis 

This  bacillus  differs  .from  typhoid  as  follows : 

1.  It  is  not  motile. 

2.  Produces  gas  in  media  containing  glucose. 

3.  Changes  the  blue  color  of  litmus  milk  media  to  a 
pink  color,  and  usually  coagulates  the  milk. 

4.  Does  not  show  the  clump  reaction. 

Agglutination  Test. — Widal  Reaction 

Take  9  drops  of  the  fluid  culture  with  a  sterile  platinum 
loop  and  place  at  separate  points  on  a  glass  slide.  Add  a 
loopful  of  patient's  serum  and  mix  all  together.  Cover 
with  a  cover  glass  and  note  the  time  (this  makes  a  dilu- 


The  Blood  117 

tion  of  TV)-  Then  take  i  loopful  of  the  TV  and  add  to 
the  4  drops  which  have  been  placed  on  the  opposite  end 
of  the  slide,  and  mix.  This  makes  a  dilution  of  ^.  Label 
and  note  the  time. 

If  before  the  end  of  I  hour  the  great  majority  of  the 
bacilli  are  both  clumped  and  motionless  in  the  TV  dilution, 
the  reaction  is  only  suggestive;  but  if  in  the  ^  dilution 


A — 9  drops  of  bouillon  culture 
12-48  hours  old. 


B — 4  drops  from  the  same  bou-       "I   I     «  o 


illon. 


o 


o 


:  • 


B 


agglutination  occurs,  it  is  practically  conclusive  evidence 
of  typhoid  infection.  It  takes  from  4  to  6  hours  for  the 
complete  change  to  occur,  and  no  less  than  six  bacteria 
must  become  agglutinated  to  constitute  a  positive  reaction. 

Hanging-drop  Slide 

Place  drops  of  oil  at  the  four  corners  of  the  cover  glass. 
Take  i  loopful  of  the  TV  or  ^V  dilution,  place  in  center 


HANGING-DROP  SLIDE 

of  the  cover  glass,  and  then  place  the  glass  drop-side 
down  over  the  depression  of  the  hanging-drop  slide. 

As  a  control,  take  3  drops  of  the  bouillon  culture  and 
place  on  a  slide  free  from  serum,  keeping  it  under  observa- 
tion during  the  experiment.  The  bacilli  must  not  clump 
and  must  remain  motile. 


n8  Clinical  Laboratory  Technic 

Detecting  B.  Typhoid  in  Small  Amounts  of  Blood.— 
Liebermann's  Method.    (Journal  A.  M.  A., 
March,  1915) 

Draw  2  drops  of  blood  into  i  c.c.  of  distilled  water. 
One  drop  represents  0.05  c.c.  of  serum  and  the  dilution 
is  equal  to  ^V 

To  this  add  I  c.c.  of  saline  and  continue  diluting  with 
different  amounts  of  saline,  making  up  to  i  c.c.  each  time. 

Koenigsfeld's  Method.    (J.  A.  M.  A.,  March,  1915) 

Two  kinds  of  medium  are  used,  Endo's  and  Drigalski- 
Conradi's ;  mannite  is  used  in  the  latter  instead  of  lactose. 
Bile  is  added  to  each  tube,  and  this,  on  top  of  the  slanting 
solid  medium,  is  inoculated  with  3  to  4  drops  of  blood. 

The  bile  has  a  tendency  to  check  the  development  of 
all  except  typhoid  bacilli.  The  colonies  develop  in  10  to 
14  hours. 

The  colon  bacillus  turns  both  tubes  red ;  acid- forming 
cocci  turn  the  Endo  media  red,  but  leaves  the  mannite 
tube  colorless. 

B.  typhosus  develops  rapidly  and  permits  an  early 
agglutination  test. 

Malarial  Parasites 

Three  species  are  associated  with  malarial  fever  in 
man. 

i.  TERTIAN  MALARIA  (Plasmodium  Vivax). — A 
small  circle  or  ring-like  body  which  stains  blue.  Within 
the  circle,  usually  near  the  circumference,  is  a  round  dot 
which  stains  red.  The  ring  becomes  larger  and  more 
irregular  in  outline,  and  pigmented  granules  appear. 
The  red  blood  cell  becomes  swollen.  The  blue  and  red 


The  Blood 


119 


elements  of  the  parasite  both  divide  into  15  to  20  seg- 
ments.    Cycle  is  completed  in  48  hours. 


Tertian  malarial  parasites  in  red  blood  corpuscles  (Wright's 
stain)  :  I,  Young  parasites  (in  the  corpuscle  on  the  right  two 
or  three  parasites)  ;  2,  young  parasites;  3,  half-grown  parasites; 
4,  half-grown  parasite  (on  the  left,  a  blood-plate  and  near  the 
center  another  blood-plate  lying  on  a  red  corpuscle)  ;  5,  half- 
grown  parasites  (in  the  corpuscle  on  the  left  two  parasites)  ; 
6,  full-grown  parasite  (the  nucleus  lies  in  a  clear  space).  All 
the  infected  blood  corpuscles  in  the  foregoing  figures  contain 
minute  granules  that  stain  red  (granular  degeneration)  (photos 
by  L.  S.  Brown). — From  "Pathologic  Technique,"  Mallory  and 
Wright. 

2.  QUARTAN  MALARIA  (Plasmodium  Malaria). — This 
form  is  much  the  same  as  the  tertian  form  in  the  early 
stages.  The  pigment  granules  are  larger  and  are  usually 


I2O 


Clinical  Laboratory  Technic 


arranged  around  the  periphery  of  the  organism;  in  the 
tertian  they  appear  to  be  distributed  all  through  it.  The 
red  blood  cells  remain  about  the  normal  size  or  a  little 
larger.  In  the  later  stages  they  become  shrunken.  They 
undergo  segmentation,  dividing  into  6  to  12  segments. 
The  full  cycle  is  completed  in  72  hours.  This  form  is 
not  common  in  America. 


i 


1  F~ 


Estivo-autumnal  malarial  parasites  in  red  blood  corpuscles 
(i,  2,  3,  and  4,  Wright's  stain)  ;  i,  2,  and  4,  young  parasites ;  3,  on 
the  left,  a  "crescent,"  on  the  concave  side  of  which  is  shown  a 
portion  of  the  periphery  of  the  red  corpuscle,  which  it  distends ; 
on  the  right,  a  young  parasite ;  5  and  7,  "  Crescents  "  in  red  blood 
corpuscles ;  6,  ovoid  form  of  parasite  in  a  red  blood  corpuscle 
(photos  by  L.  S.  Brown). — From  "Pathologic  Technique" 
Mallory  and  Wright. 


The  Blood  121 

3.  ^STIVO- AUTUMNAL  MALARIA  (Plasmodium  Fal- 
ciparum). — In  the  beginning  it  is  very  much  like  the 
two  other  forms,  only  smaller.  The  ring  becomes  larger 
and  has  the  appearance  of  a  signet  ring,  narrow  and  of 
uniform  thickness  except  at  one  section,  which  is  thicker. 
Average  number  of  segments  is  15.  The  characteristic 
"crescent"  shape  occurs  in  the  last  stages  of  this  form. 
Length  of  development  cycle  is  24  to  48  hours. 


Blood  Parasites 

(a)   Trichinella,  found  in  cases  of  trichinosis. 
(&)   Filaria  bancrofti. 

(c)  Filaria  sanguinis  hominis.  This 
parasite  is  found  in  the  blood  during 
the    night,    and    causes    dilatation    of 
the  lymphatics,  chyluria,  abscesses,  and 
elephantiasis. 

(d)  Trypanosomes,  found  in  cases 
of  sleeping  sickness. 


Wassermann  Reaction. — Obtaining  the  Blood 

The  arm  is  scrubbed  with  alcohol  2  inches  above  and 
2  inches  below  the  elbow.  Apply  the  tourniquet,  but  do 
not  obliterate  the  pulse.  Insert  the  needle  in  the  basilic 
vein  and  withdraw  5  c.c.  of  blood.  Place  this  in  the  ice 
chest  to  coagulate. 

Pipette  off  2  c.c.  of  the  clear  serum  and  incubate  at 
56°  C.  for  i  hour. 

It  has  been  found  that  if  the  blood  serum  of  a  case  of 


122  Clinical  Laboratory  Technic 

syphilis  is  mixed  in  the  presence  of  complement  with 
extracts  of  syphilitic  liver  or  alcoholic  extracts  of  normal 
organs,  the  complement  will  be  fixed  and  prevented  from 
taking  part  in  the  subsequent  hemolitic  reaction. 

Three  distinct  substances  are  required  to  form  a  com- 
plete reaction : 

1.  A  cell  to  be  destroyed  or  a  poison  to  be  neutralized. 

2.  A  substance  capable  of  destroying  the  cell  or  neutral- 
izing the  poison  (amboceptor  or  antibody). 

3.  A  completing  substance  or  the  complement,  with- 
out which  the  cell  cannot  be  destroyed  or  the  poison 
neutralized. 

Wassermann  -Test. — (Noguchi's  Serum  Diagnosis  of 

Syphilis.) — Apparatus  Needed 
6  pipettes  graduated  o.i  c.c. 
2  pipettes,  10  c.c.;  graduated  o.i  c.c. 
6  pipettes,  i  c.c. ;  graduated  o.oi  c.c. 
24  small  test  tubes,  10  x  i  cm. 

2  flasks  (100  c.c.). 

Test  tube  racks  with  parallel  rows  of  holes. 
Glass  tubing,  f-inch  bore. 

METHOD.  Place  8  c.c.  of  normal  salt  solution  in  a  flask ; 
allow  2  drops  of  blood  from  the  ear  or  finger  of  a  normal 
person  to  drop  into  the  flask  (i  drop  of  blood  to  every 
4  c.c.  of  salt  solution).  Place  in  the  refrigerator  over 
night.  The  cells  gravitate  to  the  bottom  and  are  washed 
with  an  excess  of  salt  solution.  The  supernatant  fluid  is 
poured  off  and  replaced  with  fresh  salt  solution.  Centri- 
fuge 4  times,  shaking  the  tube  after  each  addition  of  fresh 
salt  solution.  A  i%  suspension  of  washed  corpuscles 
is  used. 


The  Blood  123 

THE  TEST.  Place  i  tube  for  each  test  in  the  front  row 
and  i  tube  for  its  control  in  the  rear  row. 

Add  i  drop  of  serum  to  be  tested  (0.02  c.c.)  from  a 
capillary  pipette,  o.i  c.c.  of  40%  fresh  Guinea-pig  serum, 
made  by  adding  i  part  of  complement  to  i^  parts  of 
normal  salt  solution,  is  added  to  each  tube.  Where  fresh 
complement  cannot  be  obtained,  dried  slips  of  paper,  each 
containing  2  units  of  complement,  may  be  substituted. 
To  the  first  tube  add  the  slip  bearing  the  antigen. 

i  c.c.  of  a  i%  suspension  of  human  corpuscles  is  added 
to  both  tubes.  Shake  thoroughly  at  intervals. 

CONTROL.  To  each  tube  of  the  positive  control  add 
i  capillary  drop  of  a  syphilitic  serum  known  to  give  a 
positive  reaction. 

To  each  tube  of  the  negative  control  add  i  capillary 
drop  of  normal  serum  known  to  give  a  negative  reaction. 

Add  complement  to  each  tube. 

Antigen  only  is  placed  in  the  tubes  of  the  front  row. 

i  c.c.  of  the  corpuscle  suspension  is  now  added  to  each 
tube  and  the  rack  is  placed  in  the  incubator. 

An  hour  is  allowed  for  the  antibody  to  combine  with 
the  antigen  and  for  the  complement  to  be  fixed. 

When  the  dried  paper  is  used,  a  longer  period  of  in- 
cubation is  necessary. 

On  the  opposite  page  the  plate  shows  the  dLTerent 
stages  of  the  test. 

The  top  rack  shows: 

a.  Two  tubes  for  diagnosis. 

b.  Two  tubes  for  positive  control. 

c.  Two  tubes  for  negative  control. 

d.  Antigen. 

The  contents  of  the  tubes  are  as  follows : 


124  Clinical  Laboratory  Technic 

Front:  Test  serum  plus  complement  (2  units)  plus 
antigen  plus  corpuscle  suspension  (i  c.c.). 

Rear:  Test  serum  plus  complement  (2  units)  plus  o 
plus  corpuscles  (i  c.c.). 

Appearance  of  the  tubes  after  the  first  incubation 
(i  hour  at  37°  C). 

The  middle  rack  shows : 

d.  Antigen. 

e.  Amboceptor. 

The  two  pieces  of  paper  in  the  front  tube  represent 
antigen  and  amboceptor.  In  the  rear  tube  the  one  piece 
represents  amboceptor. 

Appearance  of  the  tubes  after  the  second  incubation, 
with  the  addition  of  the  anti-human  amboceptor  slips. 

Hemolysis  occurred  in  both  tubes  of  the  negative  set. 
In  the  positive  control  tubes  it  took  place  in  the  rear  tube 
only,  the  same  as  in  the  two  tubes  for  diagnosis ;  there- 
fore the  reaction  is  positive  and  the  serum  is  syphilitic. 

The  bottom  rack  shows : 

The  appearance  of  the  tubes  after  several  hours. 

The  absence  of  hemolysis  in  the  front  tubes  for  diag- 
nosis and  positive  control  means  positive  reaction. 

Antigen 

Free  human  heart  muscle  from  excess  of  fat  and  put 
in  meat  grinder,  and  mix  with  nine  times  its  weight  of 
cubic  centimeters  of  95%  ethyl  alcohol. 

Place  in  the  incubator  for  10  days,  shaking  the  mixture 
from  time  to  time.  Filter  and  add  0.4%  of  cholesterin. 
Return  to  the  incubator  for  3  days,  shaking  occasionally. 

When  the  cholesterin  is  all  dissolved,  the  mixture  is 
ready  for  use.  Dilute  with  4  parts  of  normal  salt  solu- 


The  Blood  125 

tion,  adding  slowly  and  in  small  quantities  at  a  time. 
The  antigen  must  not  possess  any  appreciable  hemolytic 
or  anticomplementary  qualities.  It  must  possess  suffi- 
cient antigenic  strength  to  allow  its  use  in  small  quantities. 

Noguchi  recommends  the  following  tests: 
TUBE  I 

0.4  c.c.  antigen  emulsion. 

o.i  c.c.  10%  human  corpuscles. 

Incubate  2  hours. 

Complete  absence  of  hemolysis  at  the  end  of  this  time 
indicates  that  the  antigen  emulsion  does  not  possess  any 
appreciable  hemolytic  property. 
TUBE  II 

0.4  c.c.  antigen  emulsion. 

o.i  c.c.  40%  guinea-pig  serum. 

Make  volume  i  c.c.  with  saline. 

Incubate   I   hour.     Then  add  2  units  of  amboceptor 
and  o.i  c.c.  10%  corpuscle  suspension.    Incubate  2  hours. 
Complete  hemolysis  indicates  that  the  antigen  is  not  in- 
herently anticomplementary. 
TUBE  III 

i  unit  of  syphilitic  antibody. 

0.02  c.c.  antigen  emulsion. 

o.i  c.c.  40%  guinea-pig  serum. 

Make  up  to  i  c.c.  Incubate  i  hour.  Then  add  2  units 
of  amboceptor  and  o.i  c.c.  10%  corpuscle  suspension. 
Incubate  2  hours.  Absence  of  hemolysis  shows  that 
0.02  c.c.  of  the  emulsion  has  sufficient  antigenic  strength 
to  fix  completely  2  units  of  complement.  If  the  antigen 
emulsion  fulfills  these  requirements,  it  is  suitable. 

The  amount  used  in  the  Noguchi  test  is  o.i  c.c.  (at 
least  5  units). 


JL^O  Clinical  Laboratory  Technlc 

Preparation  of  Complement  Slips 

Serum  is"  obtained  from  large  guinea  pigs.  Cut  the 
carotid  artery  and  collect  the  blood  in  a  large,  flat  dish. 
Cover  this  and  leave  at  room  temperature  for  4  hours, 
then  place  in  the  refrigerator.  Place  squares  of  thick  blot- 
ting paper  in  a  sterile,  flat  dish,  and  pour  the  serum  over 
it  until  the  paper  is  soaked  and  an  excess  remains. 

Remove  the  paper  to  another  dish  and  quickly  dry  in 
a  current  of  air  at  a  temperature  not  above  10°  C. 

Standardize  in  the  following  manner:  In  a  series  of 
tubes,  each  containing  i  c.c.  of  erythrocyte  suspension 
and  i  unit  of  amboceptor,  add  bits  of  complement  paper 
(5  mm.  width  strips)  of  increasing  length,  2,  3,  5,  7,  10, 
and  15  mm. 

Incubate  at  37°  C.  for  2  hours. 

The  tube  in  which  hemolysis  is  just  complete  contains 
i  unit  of  complement. 

The  fixation  test  requires  2  units,  therefore  the  remain- 
ing paper  is  measured  off  into  squares  having  twice  the 
dimension  of  that  bit  found  for  i  unit. 

These  slips  should  not  be  used  if  it  is  possible  to  obtain 
the  fresh  guinea-pig  serum;  and  if  the  fresh  serum  is 
used,  titrate  the  complement,  using  for.  the  test  double 
the  quantity  necessary  to  produce  complete  hemolysis  in 
i  c.c.  of  a  5%  emulsion  of  blood  cells  in  the  presence 
of  2  units  of  amboceptor. 

Amboceptor 

This  is  made  by  immunizing  rabbits  against  human 
blood  cells.  The  human  blood  corpuscles  are  collected  in 
a  sterile  centrifuge  tube.  Fill  the  tube  two-thirds  full  of 
0.85%  salt  solution.  Centrifugalize  3  minutes,  then  decant 


The  Blood  127 

the  salt  solution ;  add  more,  and  repeat  the  process  3  times. 
Increasing  amounts  are  injected  in  large  rabbits  intraperi- 
toneally  in  the  following  manner : 

ist  injection  5  c.c.  washed  human  corpuscles. 

2d          "  8  cc.,       " 

3d          "  12  c.c. 

4th         "  15  c.c. 

5th        "  20  c.c. 

Injections  are  made  at  4  or  5-day  intervals.  Nine  or 
10  days  after  last  injection  bleed  the  rabbit  from  the 
carotid  artery.  Place  the  blood  collected  at  room  temper- 
ature for  4  hours.  Collect  the  clear  serum  by  decantation 
and  leave  the  clot  for  another  24  hours.  Repeat  this  for 
3  days,  or  until  no  more  serum  is  given  out  by  the  clot. 
Mix  the  portions  of  serum  collected,  o.ooi  c.c.  of  serum 
or  less,  which  will  cause  complete  hemolysis  of  I  c.c.  of 
i%  suspension  of  human  erythrocytes  plus  0.02  c.c. 
of  guinea-pig  serum,  will  equal  I  unit. 

Amboceptor  Slips 

Cut  thin  filter  paper  (Schleicher  &  Schull,  No.  590- 
597)  into  squares,  10  x  10  cm.,  and  soak  in  the  serum; 
absorb  the  excess  with  another  sheet  of  paper.  Dry  at 
room  temperature  (place  the  squares  on  a  sheet  of  un- 
bleached muslin). 

When  thoroughly  dry,  cut  into  5  mm.  widths  and 
standardize. 

METHOD.  Take  6  tubes  and  add  i  c.c.  of  the  erythro- 
cyte  suspension,  0.02  c.c.  complement,  and  then  add  in- 
creasing lengths  of  the  amboceptor  strip,  i.  e.,  i  mm., 
2  mm.,  3  mm.,  4  mm.,  5  mm.,  6  mm.  Incubate  2  hours. 


128  Clinical  Laboratory  Technic 

The  smallest  strip  which  causes  complete  hemolysis  at 
the  end  of  the  2  hours  contains  I  amboceptor  unit. 

Mark  the  strip  in  sections  of  twice  this  length  and  cut 
off  when  doing  the  test.  Keep  the  strips  dry  and  in  a 
'sealed  receptacle. 

Definition  of  Terms 
Agglutinins 

When  bacteria  or  foreign  blood  corpuscles  are  injected 
Jnto  an  animal,  a  new  property  is  developed  in  the  serum 
of  that  animal ;  and  this  serum,  when  deprived  of  its 
own  complement,  by  inactivation  or  dilution,  is  capable 
of  clumping  the  bacteria  or  corpuscles  used  for  im- 
munization. 

n 
Amboceptor 

One  of  two  active  principles  necessary  to  cause  hemol- 
ysis, bacteriolysis,  or  any  cytolysis  caused  by  serum,  the 
other  active  principle  being  complement. 

Antibodies 

Immune  body  is  a  synonym  of  antibody.  Antibodies 
possess  specific  affinity  for  the  antigens  which  are  used 
for  their  production.  A  group  of  antibodies  is  capable 
of  producing  antibodies  when  injected  into  another  animal, 
thus  forming  anti-antibodies. 

Anti-complementary  Action 

Substances  capable  of  reducing  or  removing  the  action 
of  the  complement. 

Antigen 

Substances  which  have  the  power  of  producing  specific 
antibodies,  as  bacteria  and  blood  corpuscles,  which  are 


The  Blood  129 

antigens  because  they  produce  specific  antibodies  called 
amboceptors  and  agglutinins.  Diphtheria  toxin  is  an  anti- 
gen, and  injections,  of  it  are  followed  by  a  specific  anti- 
toxin. 

Complement 

One  of  the  two  active  principles  necessary  for  hemol- 
ysis.  The  other  principle  is  called  amboceptor,  and  is 
unable  to  cause  a  dissolution  of  cells  without  the  first; 
hence  the  term  complement. 

Cytolysis 

Dissolution  of  cells  by  specific  amboceptors  and  com- 
plement. 

Hemolysis 

Dissolution  of  blood  corpuscles  by  various  forces,  set- 
ting the  hemoglobin  free  into  the  medium  in  which  cor- 
puscles are  suspended.  Distilled  water  will  cause  hemol- 
ysis,  also  acid  and  alkalies.  Hemolysis  by  serum  is  some- 
what different,  and  is  caused  by  two  distinct  groups  of 
substances,  complement  and  amboceptor,  both  contained 
in  the  serum,  and  the  one  is  inactive  without  the  other. 

Immune  Bodies 

Synonymous  with  antibodies. 

Inactivation 

When  fresh  serum,  which  contains  both  amboceptor  and 
complement,  is  heated  at  55°  for  ^  hour,  it  becomes  in- 
active, because  of  the  destruction  of  the  complement.  The 
amboceptor  is  not  affected  materially  by  the  process. 


130 


Clinical  Laboratory  Technic 


Blood  Pressure 

Pressure  is  generally  applied  to  the  brachial  artery  by 
means  of  the  arm  band. 

The  blood  pressure  is  variable,  especially  that  in  the 
arteries.  This  pressure  is  expressed  as  being  equal  to  so 


"Tvcos"  BLOOD  PRESSURE 
APPARATUS 

The  gauge  measures  pressure  from  o  to  300  millimeters  of 
mercury  and  the  dial  is  movable.  The  needle  should  be  adjusted 
at  the  o  mark  before  taking  the  pressure.  The  dial  is  graduated 
to  read  in  millimeters  and  fifths  of  millimeters. 

many  millimeters  of  mercury,  and  by  this  expression  is 
meant  that  the  pressure  within  the  artery  is  able  to  sup- 
port a  column  of  mercury  that  many  millimeters  in  height. 

Systolic  Pressure 

The  systolic  pressure  is  the  maximum  pressure  caused 
by  the  systole  of  the  heart,  or  the  apex  of  the  pulse  wave. 

Diastolic  Pressure 

This  is  the  minimum  pressure  in  the  artery — the 
pressure  at  the  end  of  the  diastole  of  the  heart  or  at  the 
bottom  of  the  pulse  wave. 

Under  normal  conditions  the  systolic  pressure  in  the 
adult,  expressed  in  terms  of  a  mercury  column,  equal 


The  Blood  131 

110-115  millimeters,  while  the  diastolic  pressure  is  only 
65-75  millimeters.  Maximum  pressure  during  the  first 
years  of  life  varies  from  75-90  mm.  Hg.  The  pulse  pres- 
sure is  the  difference  between  the  systolic  and  diastolic 
pressure.  The  pulse  pressure  in  the  artery  averages 
45  millimeters  of  mercury. 

The  mean  pressure  is  obtained  by  dividing  the  sum  of 
the  systolic  and  diastolic  pressure  by  2,  or  by  adding  half 
of  the  pulse  pressure  to  the  diastolic  pressure.  A  blood 
pressure  determination  should  be  a  routine  procedure  in 
the  first  examination  of  every  patient. 

Palpatory  Method 

Find  the  pulse  at  the  wrist  of  the  arm  to  which  the  arm 
band  has  been  applied,  and  while  the  pulse  is  under  obser- 
vation raise  the  pressure  with  the  hand  bellows  or  pump 
until  the  pressure  within  the  constricting  band  is  sufficient 
to  prevent  the  im-pulse  from  reaching  the  wrist.  The 
position  of  the  arrow  on  the  face  of  the  dial  at  the  instant 
when  the  pulse  passes  the  compressing  band  will  repre- 
sent the  systolic  pressure.  Readings  should  be  made  at  the 
return  of  the  full  pulse. 

Auscultatory  Method 

This  method  is  used  where  there  is  no  diastolic  fluctua- 
tions. Instead  of  feeling  the  pulse,  a  pulse  tone  caused 
by  pressure  of  the  constricting  cuff  is  listened  to  through 
the  stethoscope.  Raise  the  pressure  to  the  obliteration 
of  the  pulse,  then  place  the  stethoscope  over  the  brachial 
artery  below  the  cuff.  The  pressure  is  gradually  allowed 
to  fall  and  a  pulse  tone  is  heard  as  the  circulation  com- 
mences, and  this  tone  undergoes  a  number  of  changes 


132  Clinical  Laboratory  Technic 

p 

until  it  becomes  very  faint  and  almost  disappears.    The 

reading  of  the  sphygmomanometer  at  this  moment  repre- 
sents the  diastolic  pressure. 

Factors  Influencing  Blood  Pressure 

Posture :  Standing  Sitting  Supine  Rt.  Lateral  L.  Lateral 
132  134          152  155  no 

Age :  During  the  first  years  it  varies :  75-90. 
15  years  to  21 :  100-115. 
Adults :  120-140. 

Sex :  Female  sex  is  lower  as  a  rule. 

Time  of  day:  In  the  early  hours  of  sleep  there  is  a  de- 
cided fall,  which  gradually  rises  towards  morning. 
Minimum — A.M.  Maximum — P.M. 

Exercise:  Raises  pressure,  also  emotion  and  excitement. 

Clinical  value:  In  the  treatment  of  children,  as  a  guide 
to  stimulation  and  other  treatment;  in  obstetrics,  in 
diagnosis  and  treatment  of  toxemias;  also  in  myo- 
carditis, nephritis,  typhoid  fever,  pneumonia,  and 
arterio-sclerosis. 

Blood  Chemistry 

Blood  chemical  analysis  surpasses  in  value  qualitative 
and  quantitative  urinary  analysis. 

It  is  preferable  to  take  the  blood  in  the  morning  before 
breakfast.  (See  page  138,  method  of  collecting.) 

Determination  of  Non-Protein  Nitrogen. — Folin's 
Method 

Normally  25  to  30  milligrams  of  non-protein  N  in 
100  c.c.  blood.  This  is  increased  in  chronic  nephritis, 
uremia,  plumbism,  acute  intestinal  obstruction,  and  pro- 
static  disease. 


The  Blood  133 

Solutions  Needed 

i.  ACID  DIGESTION  MIXTURE.  Mix  300  c.c.  of  phos- 
phoric acid  with  100  c.c.  concentrated  H2SO4.  Cover 
and  set  aside  for  several  days  (not  absolutely  necessary). 
To  100  c.c.  of  clear  acid  add  10  c.c.  of  6%  CuSO4 
solution  and  100  c.c.  ammonia  free  water.  Use  I  c.c.  in 
the  non-protein  nitrogen  digestion. 

2.  NESSLER'S     SOLUTION     (Stock     Solution).       Mix 
150  grams  K.  I.,  no  grams  iodine,  and  100  c.c.  water; 
add  an  excess  of  metallic  mercury  (140  to  150  grams). 
Shake  vigorously  for  15  minutes.    The  solution  becomes 
quite  hot.     When  the  red  iodine  solution  has  begun  to 
pale,  cool  in  running  water  and  continue  shaking  until 
the  reddish  color  is  replaced  by  the  greenish  color  of  the 
double  iodide.     Separate  the  solution  from  the  surplus 
mercury    by    decantation    and    washing    with    distilled 
water.    Dilute  to  2  liters.     To  75  c.c.  of  this  stock  solu- 
tion, add  75  c.c.  of  distilled  water  and  350  c.c.  of  10% 
NaOH  solution. 

3.  AMMONIUM  SULPHATE  FOR  NITROGEN  STANDARD. 
0.4716  gram  dissolved  in  I  liter  of  ammonia-free,  dis- 
tilled water. 

Ammonia- free  water  is  obtained  from  ordinary  dis- 
tilled water  by  the  addition  of  a  little  bromine  water  and 
a  few  drops  of  concentrated  sodium  hydroxide. 

The  standard  most  commonly  required  is  0.3  milligram 
of  N.  Add  3  c.c.  of  the  standard  ammonium  sulphate 
solution  (containing  i  milligram  of  N  per  10  c.c.)  to  a 
100  c.c.  flask.  Add  2  c.c.  of  the  phosphoric-sulphuric  acid 
mixture.  Dilute  to  about  60  c.c.  and  add  30  c.c.  of 
Nessler's  Solution.  The  unknown  and  the  standard 
should  be  Nesslerized  simultaneously. 


134  Clinical  Laboratory  Technic 

METHOD.  Transfer  5  c.c.  of  protein  free  filtrate  to 
a  dry  75  c.c.  Pyrex  test  tube  graduated  at  35  and  50  c.c. 
Add  i  c.c.  of  sulphuric-phosphoric  acid  mixture.  Add 
a  dry  quartz  pebble  or  a  piece  of  platinum.  Boil  vigor- 
ously over  a  microburner  until  SO3  fumes  form  (4-7 
minutes).  Turn  down  flame  to  just  visible  boiling,  cover 
mouth  of  test  tubes  with  watch  glass.  Continue  heating 
gently  until  clear  solution  results.  Allow  to  cool  70-90 
seconds.  Add  15  to  25  c.c.  distilled  water.  Cool  arid 
add  water  to  35  c.c.  mark,  then  add  Nessler's  Solution 
to  the  50  c.c.  mark.  Insert  clean  rubber  stopper,  mix, 
and  compare  with  the  standard  in  colorimeter. 

STANDARD.  3  c.c.  standard  ammonium  sulphate  solu- 
tion in  100  c.c.  flask.  Add  2  c.c.  of  sulphuric-phosphoric 
acid  mixture,  50  c.c.  water,  30  c.c.  of  Nessler's  Reagent, 
and  fill  to  the  mark  with  distilled  water. 

CALCULATION.  Reading  of  the  standard  divided  by 
reading  of  the  unknown,  multiplied  by  30  equals  mg. 
non-protein  N  per  100  c.c.  blood. 

The  unknown  and  the  standard  should  be  Nesslerized 
simultaneously.  If  the  standard  is  set  at  20  millimeters 
for  the  color  comparison,  20  divided  by  the  reading  and 
multiplied  by  0.3  gives  the  non-protein  nitrogen  in  i  c.c. 
of  blood,  because  0.5  c.c.  (the  amount  of  blood  repre- 
sented in  5  c.c.  of  the  blood  filtrate)  Nesslerized  at  a 
volume  of  50  c.c.  is  equivalent  to  I  c.c.  Nesslerized  at 
a  volume  of  100  c.c.  The  non-protein  nitrogen  per 
100  c.c.  of  blood  is,  therefore,  20  divided  by  the  reading 
and  multiplied  by  30  (0.3  X  100). 
Blood  Urea.  Folin's  Method.  —  Solutions  Needed 

UREASE  SOLUTION.  Wash  3  grams  of  permutit  in  a 
flask  once  with  2%  acetic  acid,  then  twice  with  water. 


The  Blood  135 

Add  5  grams  of  fine  Jack  bean  meal  and  100  c.c.  of 
15%  alcohol.  Shake  gently  but  continuously  for  15 
minutes.  Pour  on  a  large  filter  and  cover  with  a  watch 
glass.  The  filtrate  contains  the  urease,  and  this  solu- 
tion will  keep  4  to  6  weeks  in  an  ice  box. 

BUFFER  MIXTURE.  Dissolve  69  grams  of  monosodium 
phosphate  and  179  grams  of  crystallized  disodium  phos- 
phate in  800  c.c.  of  warm,  distilled  water.  Cool  and 
dilute  to  a  liter. 

METHOD.  Transfer  5  c.c.  of  tungstic  acid  blood  filtrate 
to  a  Pyrex  ignition  tube  (200  X  25  mm.).  If  the  Pyrex 
tube  has  contained  Nessler's  Solution,  rinse  with  nitric 
acid  and  then  wash  with  water  before  introducing  the 
blood  filtrate. 

Add  2  drops  of  buffer  mixture  and  i  c.c.  of  urease 
solution.  Immerse  tube  in  warm  water,  40  to  50°  C., 
and  leave  for  5  minutes.  The  ammonia  formed  from  the 
urea  is  obtained  by  distillation,  using  a  test  tube  gradu- 
ated at  25  c.c.  and  containing  2  c.c.  of  0.05  N  hydro- 
chloric acid  as  a  receiver  (see  illustration,  page  136). 

Add  a  dry  pebble  to  the  urease  blood  filtrate,  2  drops 
of  paraffin  oil,  and  2  c.c.  of  saturated  borax  solution. 
Insert  firmly  the  rubber  stopper  carrying  the  delivery 
tube  and  receiver. 

Boil  at  a  moderately  fast,  uniform  rate  for  4  minutes. 
Do  not  cut  down  the  flame  during  the  distillation.  The 
boiling  should  not  be  so  brisk  that  the  emission  of  steam 
from  the  receiver  begins  before  the  end  of  3  minutes. 
At  the  end  of  4  minutes  slip  off  the  receiver  from  the 
rubber  stopper  and  let  it  rest  in  a  slanting  position  while 
the  distillation  is  continued  for  i  minute.  Rinse  the 
lower  end  of  the  delivery  tube  with  a  little  water.  Cool 


i36 


Clinical  Laboratory  Tec Jin ic 


the  distillate  with  running  water.  Dilute  to  20  c.c. 
Transfer  3  c.c.  of  standard  ammonium  sulphate  solution 
to  a  100  c.c.  flask  and  dilute  to  75  c.c.  Nesslerize,  using 
10  c.c.  of  Xessler's  Solution  for  the  standard,  and  2.5  c.c. 


A,  At  beginning.  B,  Toward  end  of  distillation. — From  Folin's 
"Laboratory  Manual  of  Biologic  Chemistry."   D.  Appleton  &  Co. 


The  Blood  137 

for  the  unknown  in  the  test  tube.    Dilute  both  to  volume 
and  make  color  comparison. 

CALCULATION.  Divide  20  by  the  colorimetric  reading 
and  multiply  by  I5.1  This  gives  the  urea  nitrogen  in 
milligrams  per  100  c.c.  of  blood. 

Directions  for  Using  the  Duboscq 

One  glass  cylinder  contains  the  Standard  "Known," 
and  the  other  contains  the  "Unknown"  (the  solution  to 
be  studied).  Regulate  the  mirror  so  that  by  looking 
through  the  eyepiece  the  two  halves  of  the  field  of 
vision  appear  of  equal  intensity ;  the  cups  must  be  empty 
and  clean.  Then  pour  the  solutions  into  the  cylinders. 
By  raising  or  lowering  the  cylinder  of  the  "Unknown/' 
the  two  halves  of  the  field  can  be  brought  to  an  identical 
intensity.  Read  on  the  scale  the  heights  of  the  two 
layers  of  liquid.  The  standard  "Known"  is  usually  set 
at  20  millimeters. 

Determination    of    Sugar    in    Blood.      Folin's    New 
Method. — Solutions  Needed 

1.  SATURATED  SODIUM  CARBONATE  SOLUTION. 

2.  STANDARD  SUGAR  SOLUTION.     Dissolve  i  gram  of 
pure  anhydrous  dextrose  in  water  and  dilute  to  100  c.c. 
Mix,  add  four  drops  of  xylene  or  toluene,  and  bottle. 
Dilute  5  c.c.  to  500  c.c.,  giving  a  solution  10  c.c.  of  which 
contains  i  milligram  of  dextrose.      Add   few  drops  of 
xylene  for  preservative.    Use  2  c.c.  for  each  determina- 
tion. 

3.  ALKALINE  COPPER  SOLUTION.     Dissolve  40  grams 

1  Nesslerized  at  25  c.c.  Therefore  the  reading  represents  only 
one-half  as  much  nitrogen  as  in  the  non-protein  nitrogen  deter- 
mination. 


138  Clinical  Laboratory  Technic 

of  anhydrous  sodium  carbonate  in  400  c.c.  of  water.  Add 
7.5  grams  tartaric  acid  and  when  the  latter  has  dissolved, 
add  4.5  grams  of  crystallized  copper  sulphate.  Mix,  and 
make  up  to  I  liter. 

4.  PHOSPHOTUNGSTIC-PHOSPHOMOLYBDIC  ACID.  To 
25  grams  of  molybdenum  trioxide  (MoO3),  or  34  grams 
of  ammonium  molybdate  (NH4)2  (MoO4),  add  140  c.c. 
of  10%  sodium  hydroxide  and  150  c.c.  of  distilled  water. 
Boil  20  minutes  to  drive  off  ammonia.  Then  add  100 
grams  sodium  tungstate,  50  c.c  of  85%  phosphoric  acid, 
100  c.c.  of  concentrated  hydrochloric  acid,  and  dilute  to 
a  volume  of  700  to  800  c.c.  Close  the  mouth  of  the  flask 
with  a  funnel  and  watch  glass.  Boil  gently  for  4  hours, 
adding  hot  water  from  time  to  time  to  replace  that  lost 
during  boiling.  Cool,  dilute  to  I  liter.  This  is  diluted 
when  used  in  connection  with  the  determination  of  blood 
sugar  (100  c.c.  of  reagent  with  50  c.c.  water  and  50  c.c. 
of  concentrated  hydrochloric  acid). 

METHOD.  Collect  the  blood  over  finely  powdered  po- 
tassium oxalate  (20  milligrams  for  10  c.c.  blood).  Meas- 
ure 10  c.c.  into  a  200  c.c.  flask.  Add  70  c.c.  of  water, 
mix.  Add  10  c.c.  of  10%  sodium  tungstate  and  mix. 
With  a  graduated  pipette  or  burette,  add  slowly,  and  with 
shaking,  10  c.c.  of  %  normal  sulphuric  acid  (35  grams 
concentrated  sulphuric  acid,  chemically  pure,  diluted 
to  i  liter).  Close  the  mouth  of  the  flask  with  a  rubber 
stopper,  and  shake.  Filter  through  dry  filter.  Heat  a 
beaker  of  water  to  vigorous  boiling.  Transfer  2  c.c.  of 
tungstic  acid  blood  filtrate  to  a  test  tube  (20  mm.  X 
200  mm.)  graduated  at  25  c.c.  Into  two  other  similar 
tubes  put  2  c.c.  standard  sugar  solution  containing  re- 
spectively 0.2  and  0.4  milligrams  of  dextrose.  Add  to 


The  Blood  139 

each  tube  2  c.c.  of  the  alkaline  copper  tartrate  solution. 
Heat  in  the  boiling  water  6  minutes.  Remove  the  test 
tubes  and  add  at  once  i  c.c.  of  the  diluted  phosphotung- 
stic-phosphomolybdic  acid  solution.  Mix,  cool,  and  add 
5  c.c.  of  saturated  sodium  carbonate  solution.  Dilute  to 
25  c.c.  mark.  After  5  minutes  make  color  comparison. 
The  depth  of  the  standard  (in  millimeters)  multiplied 
by  100  (weaker  solution)  or  200  (stronger  solution) 
divided  by  the  reading  of  the  unknown  gives  the  sugar 
content,  in  milligrams,  per  100  c.c.  blood.  Normally 
about  0.1%  sugar  in  blood.  100  milligrams  is  the  same 
as  o.i  (i  decigram),  therefore  100  milligrams  per  100 
c.c.  and  0.1%  are  the  same. 

REFERENCES:  Hammarsten's  Physiological  Chemistry. 
Mallory's  Principles  of  Pathologic  Histology.  Schleip's 
Atlas  of  Hematology.  Gradwohl's  .Blood  and  Urine 
Chemistry. 


140  Clinical  Laboratory  Technic 


Blood 

Name Date. 

Specimen  taken  from 

Hemoglobin : 

Examination  of  Stained  Specimen. 

Red  Corpuscles 

Volume  Index 

Anisocytosis    

Poikilocytosis    

Achromia    

Polychromatophilia    

Stippling   

Nucleated  Corpuscles  

Normoblasts    

Megaloblasts    


White  Corpuscles :  Differential  Count.    Cells  Counted : 

Polymorphonuclear  Neutrophiles  % 

Lymphocytes    % 

Large  Mononuclears % 

Eosinophiles % 

Mast  Cells    % 

Myelocytes,  Neutrophilic % 

Eosinophilic  % 

Basophilic % 


Number  of  Red  Corpuscles  per  cmm 

"   White         "         " 

Parasites. 
Color  Index. 
Blood  Plates. 

REMARKS  : 

Signed 


CHAPTER  VIII 

BACTERIA 

Form 

The  form  of  bacteria  is  very  simple.  They  are  either 
spheres,  straight  rods,  or  bent  rods  (spiral). 

They  are  known  as  cocci  or  micrococci  in  spherical 
form ;  the  straight  rods  are  called  bacilli ;  and  the  bent 
rods,  spirilla. 

1  234 

56  7  89 


10  11  12 


VARIOUS  FORMS  OF  BACTERIA 

i.  Staphylococci.  2.  Streptococci.  3.  Diplococcus.  4.  Tetrad. 
5.  Bacilli.  6.  Bacilli  in  chains.  7.  Bacilli  forming  spores.  8. 
Bacillus — drumstick  form.  9.  Bacillus  with  polar  bodies.  10. 
Spirilla,  n.  Spirochetae.  12.  Involution  forms  (degenerate). 

Chemical  Composition 

The  composition  varies  with  the  species  and  nature  of 
the  culture  media.  They  consist  mostly  of  water,  80  to 
88%,  and  varying  amounts  of  salts,  fats,  and  other  albu- 
minous substances. 

141 


'142  Clinical  Laboratory  Technic 

Size 

The  unit  of  measurement  in  microscopy  is  the  micron 
(/x),  or  micromillimeter.  This  is  .001  of  a  millimeter,  or 
approximately  ^^QQ  of  an  inch. 

Micrococci,  bacilli,  and  spirilla  average  about  i  micron. 

Color 

Brilliant  coloring  is  displayed  by  many  bacterial 
growths. 

Rose-red  )  0 

I  Sarcinae. 

Orange-yellow  J 

Red:  B.  Prodigiosus  deposits  a  moist  red  material  on 
bread  and  other  articles  of  food. 

Green  1  B.  Pyocyaneus :  Green  fluorescence  ;  gives  color 

Blue    J         to  pus. 

Phosphorescence:    Bacteria  in  the  ocean  and  fish. 

Ferments 

Fermentation  is  the  formation  of  useful  products  by 
the  action  of  bacteria.  Ferments  dissolve  protein  sub- 
stances, form  sugar  from  starch,  change  cane  sugar  into 
glucose,  decompose  fat  into  fatty  acids  and  glycerin,  and 
convert  ammonia  into  nitrous  acid,  which  forms  nitrites ; 
these  nitrites  are  changed  to  nitric  acid,  then  to  nitrates. 

Acid 

The  acids  commonly  resulting  from  bacterial  growth 
are  lactic,  acetic,  oxalic,  formic,  and  hippuric. 

Gas 

The  putrefactive  action  of  bacteria  forms  hydrogen 
sulphide,  nitrogen,  carbon  dioxide,  and  other  gases. 


Bacteria  143 

Gas  formation  can  be  observed  by  making  stab  cultures 
in  agar  containing  the  fermentable  nutrient  substances. 

Bacteria  can  be  studied  microscopically,  in  the  living 
and  unstained,  by  means  of  the  "hanging  drop" 
preparation. 

METHOD.  Transfer  a  drop  of  fluid  media  in  which 
bacteria  have  been  growing  to  a  cover  glass  ;  or  if  the  bac- 
teria have  been  growing  in  solid  media,  make  an  emulsion 
with  i  drop  of  bouillon  or  distilled  water.  Smear  the  four 
sides  of  the  cover  glass  with  balsam  or  vaseline,  and  invert 
over  the  slide  so  that  the  drop  hangs  freely  within  the 
hollow  space.  The  movements,  development,  and  multi- 
plication of  the  bacteria  may  be  easily  observed. 

Nakanishi's  Method 

Cover  a  slide  with  an  aqueous  solution  of  methylene 
blue.  Dry,  then  place  the  emulsified  bacteria  on  a  cover 
slip,  and  drop  face  downward  upon  the  blue  background 
of  the  slide.  Cytoplasm  is  stained  blue  and  nuclear 
material  is  stained  a  reddish  color. 

Movement 

Progressive  (in  some  cases  the  movement  is  vibratory). 
Bacteria  move  back  and  forth,  but  do  not  change  their 
relative  positions,  and  this  is  known  as  the  Brownian 
movement. 

Only  a  few  of  the  micrococci  are  motile.  Many  of  the 
bacilli  and  spirilla  are  motile,  and  this  movement  is  a 
change  of  position  caused  by  certain  protoplasmic  proc- 
esses which  these  bacteria  possess,  known  as  cilia  or 
flagella.  The  cilium  has  a  simple  curve,  while  the  flagel- 
lum  has  a  compound  curve. 


144  Clinical  Laboratory  Technic 

Spores 

Bacilli  and  spirilla  form  spores  within  the  cell,  and  these 
are  called  endospores. 

Examination  by  Cultures 

The  material  to  be  examined  is  obtained  by  means  of 
"swabs,"  and  these  swabs  are  made  with  a  piece  of  rattan 
or  wire  about  15  cm.  in  length,  on  one  end  of  which  is 
firmly  twisted  a  small  pledget  of  absorbent  cotton.  Place 
this  in  a  test  tube,  cotton  end  first ;  stopper  the  tube  with 
cotton  and  sterilize  \  hour  at  180°  C. 

METHOD.  Remove  the  stopper,  taking  care  to  keep  the 
end  which  enters  the  tube  sterile.  Bring  the  cotton  end 
of  the  swab  in  contact  with  the  material  to  be  examined ; 
avoid  touching  anything  else.  Replace  the  swab  in  the 
tube  and  insert  the  stopper.  Take  the  tube  of  medium 
which  is  to  be  inoculated  and  pass  the  stopper  through 
the  flame.  Hold  the  two  tubes  in  the  left  hand,  between 
the  thumb  and  first  finger,  in  a  slanting  position.  Re- 
move the  stoppers  and  hold  between  the  fingers  of  the 
right  hand,  and  inoculate  the  surface  of  the  medium  by 
gently  rubbing  the  swab  over  it.  Flame  the  end  of  the 
tube  of  medium,  replace  the  stopper,  flame  again,  incu- 
bate 12  to  24  hours.  Smear  and  stain. 

The  majority  of  bacteria  require  oxygen  for  their 
growth,  but  some  fail  to  grow  unless  it  is  excluded. 

Aerobes 

"Obligatory  aerobes"  requiring  oxygenated  environ- 
ment as  a  necessary  condition  for  growth  are:  gono- 
coccus,  bacillus  influenza,  bacillus  pestis. 


Bacteria  145 

Anaerobes 

"  Obligatory  anaerobes  "  which  develop  only  in  an  en- 
vironment from  which  free  oxygen  has  been  excluded 
are  the  bacillus  tetanus,  bacillus  malignant  edema,  bacil- 
lus anthrax,  bacillus  aerogenes  capsulatus,  and  bacillus 
botulinus. 

"Facultative  anaerobes"  are  numerous  parasitic  and 
saprophytic  bacteria  which  develop  and  multiply  both 
under  aerobic  and  anaerobic  conditions. 

Pasteur's  Method  for  Cultivating  Anaerobes 

Add  2  c.c.  of  sterile  albolene,  oil,  or  paraffin  to  the  test 
tube  of  media.  Cool  the  lower  portion  of  the  media  to 
40°  C.  while  leaving  the  paraffin  fluid,  and  inoculate  the 
media. 

Wright's  Method 

1.  Inoculate  culture  media  in  test  tube. 

2.  Place  the  absorbent  cotton  stopper  in  until  it  lies 
15  mm.  below  the  mouth  of  the  tube. 

3.  Fill  space  above  with  dry  pyrogallic  acid. 

4.  Pour  sufficient  strong  solution  of  sodium  hydrate 
( 10%  )  to  dissolve  the  pyrogallic  acid.   This  must  be  done 
quickly.     Insert  rubber  stopper  in  tube  so  as  to  close  it 
tightly. 

Bacterial  Vaccines 

1.  Isolate  the  organism  and  inoculate  three  or  four 
"slants"  of  agar  culture  media.    Incubate  12  to  24  hours. 
Then  add  z  or  2  c.c.  of  sterile  0.9%  saline  solution  to 
each  tube  of  culture  media,  and  gently  rub  off  the  growth 
with  a  sterile  platinum  loop. 

2.  Transfer  the  bacterial  suspension  to  a  sterile  test 
tube. 


146  Clinical  Laboratory  Technic 

t  3.  Heat  the  end  of  the  test  tube  containing  the  emul- 
sion, and  also  a  piece  of  glass  tubing,  then  join  these  two, 
ends  and  draw  out  the  test  tube  about  I  inch  from  the 
mouth  in  a  blowpipe  flame.  Draw  this  out,  cool,  file 
the  constricted  portion,  and  seal  in  the  flame. 

4.  Shake  vigorously. 

5.  Place  the  tube  in  a  water  bath  at  60°   C.    Heat 
i  hour. 

6.  Shake,  then  break  open,  and  take  a  few  drops  for 
cultures   and  standardization,    after   which   the   tube   is 
again  sealed. 

Method  of  Standardization 

Draw  up  the  bacterial  suspension  to  the  mark  0.5  in 
the  red  blood  corpuscle  pipette.  Dilute  1-200  with  dis- 
tilled water.  Place  a  small  drop  on  the  center  of  a  0.02 
millimeter  counting  chamber.  Count  with  the  high  power 
objective.  After  10  minutes  begin  the  count. 

EXAMPLE. 

Number  of  bacteria  counted  X  the  dilution  X  20,000 

100  (number  of  squares  counted) 

equals  the  number  of  bacteria  in  I  cubic  millimeter.  Mul- 
tiply the  result  by  1,000  to  obtain  the  number  of  bacteria 
in  i  cubic  centimeter. 

After  heating  the  suspension,  break  the  small  end  of 
the  tube  and  make  a  "plant"  of  the  emulsion  upon  the 
surface  of  a  blood  serum  "slant"  to  test  the  sterility  of 
the  emulsion. 

Preparing  a  Dilute  Vaccine  for  Injection 

Place  50  c.c.  of  a  0.9%  salt  solution  in  a  flask,  stopper 
with  a  rubber  nipple,  and  sterilize. 


Bacteria  147 

If  it  is  desired  to  have  a  vaccine  containing  200,000,000 
bacteria  per  cubic  centimeter  and  the  stock  vaccine 
showed  5,000,000,000  bacteria  per  cubic  centimeter,  it 
would  be  necessary  to  draw  out  2  c.c.  of  the  salt  solution 
with  a  sterile  syringe  needle  inserted  through  the  rubber 
cap  and  replace  it  with  2  c.c.  of  the  bacterial  suspension. 

EXAMPLE.  In  introducing  2  c.c.  of  a  vaccine  contain- 
ing 5,000,000,000  bacteria  per  cubic  centimeter  we  throw 
10,000,000,000  bacteria  in  a  volume  equal  to  50  c.c. 

Then  each  cubic  centimeter  of  the  50  c.c.  in  the  flask 
would  contain  10,000,000,000  divided  by  50,  or  200,- 
000,000  in  each  cubic  centimeter. 

Add  l/4%  of  trikresol  to  the  vaccine  to  insure  sterility. 
Minimum  and  Maximum  Doses  Expressed  in  Mil- 
lions (Wilson) 

Staphylococcus,  150  and  900. 

Streptococcus,  6  and  68. 

Gonococcus,  45  and  900. 

Meningococcus,  300  and  900. 

B.  typhosus  (treatment),  100  and  250. 

B.  typhosus  (prophylaxis),  500  and  1,000. 

B.  pneumonise,  44. 

B.  tuberculosis,  1/20,000  to  200  milligrams. 
Lipo-Vaccines 

The  bacteria  are  suspended  in  oil  instead  of  salt  solu- 
tion.   Absorption  is  slower,  the  reaction  less,  and  massive 
doses  can  be  given  at  one  injection. 
The  Plate  Method  of  Petri  (Isolating  Bacteria) 

The  Petri  plates  consist  of  two  circular  glass  dishes; 
the  larger  is  used  as  a  cover.  Before  using,  these  plates 
are  wrapped  in  paper  and  sterilized  |  hour  at  180°  C. 


148  Clinical  Laboratory  Technic 

.Melt  2  or  4  tubes  of  agar-agar  or  gelatin,  cool  to 
40°  to  42°  C.,  then  with  a  sterile  platinum  loop  introduce 
a  loopful  of  infected  material  into  the  first  tube,  and  mix 
thoroughly.  Four  loopfuls  of  this  are  taken  and  mixed 
with  the  second  tube,  etc.  Pour  the  contents  of  the  2  or 
4  tubes  on  separate  Petri  dishes,  cover,  label  the  dilution, 
and  incubate. 

When  agar-agar  is  used,  incubate  24  hours  at  37°  C. ; 
but  when  gelatin  is  used,  it  should  be  set  in  a  cool  place 
to  solidify,  and  then  in  a  place  free  from  dust,  at  room 
temperature,  for  several  days. 

A  pure  culture  or  one  variety  of  bacteria  is  obtained 
in  this  way,  as  each  organism  develops  from  a  colony  of 
its  own  kind,  in  an  area  somewhat  isolated. 


Isolation  and  Identification  of  Streptococcus 
Hemolyticus 

Inoculate  fluid  blood  agar  (at  45°  C.)  and  pour  into 
sterile  Petri  dishes.  Incubate  at  37°  C.  for  18  to  24 
hours.  Examine  plates  for  isolated  colonies  with  well- 
defined  colorless  zones  of  hemolysis. 

1.  Transfer  a  typical  isolated  hemolytic  colony  to  a 
tube  of  bouillon.     Incubate   12  hours.     Make  a  smear, 
stain  by  Gram's  method.     Note  length  of  chains,  size, 
shape  arrangement  of  cocci  in  chains. 

2.  Mix  0.5  c.c.  of  a  5  per  cent  suspension  of  washed 
rabbit  blood  corpuscles  in  physiological  salt  solution.    In- 
cubate in  a  water  bath  at  37°  C.  for  two  hours. 

Hemolytic  pathogenic  streptococci  of  human  origin 
produce  laking  of  blood  under  these  conditions. 


Bacteria  149 

3.  To  i  c.c.  of  bouillon  culture  add  one-fifth  volume 
of  sterile  ox  bile.  Incubate  one  hour.  Pneumococci  may 
cause  some  hemolysis  on  blood  agar,  but  solubility  in  bile 
distinguish  them  from  streptococci. 

Bacteria  of  Pathogenic  Significance  Commonly 
Encountered  in  Pathological  Processes  in  Man 

1.  STAPHYLOCOCCI  PYOGENES  AUREUS:  Small  cocci  ar- 
ranged in  masses  or  clumps.    Stained  dark  blue  by  Gram's 
method.    The  colonies  on  culture  media  are  golden  yellow 
in  color. 

Staphylococcus  albus  and  staphylococcus  pyogenes 
citreus  differ  from  staphylococcus  pyogenes  aureus  in 
color  of  their  colonies,  and  they  are  much  less  pathogenic. 

2.  STREPTOCOCCI  PYOGENES:  Small  cocci  arranged  in 
chains.    The  growth  on  culture  media  occurs  in  minute, 
grayish,    translucent    colonies.     Stained    dark    blue    by 
Gram's  method. 

3.  PNEUMOCOCCI:  Oval,  lancet-shaped  organisms  ar- 
ranged in  pairs.    In  pus  and  blood  the  organism  is  in- 
vested with  a  hyaline  zone  called  the  capsule.     Stained 
dark  blue  by  Gram's  method.    The  colonies  appear  minute 
and  colorless  on  culture  media,  resembling  drops  of  dew. 

4.  BACILLI   COLI   COMMUNIS:   Medium-sized  bacillus 
with    rounded   ends,    sometimes    short   and   coccus-like. 
Decolorized  by  Gram's  method.    Growth  on  culture  media 
appears  as  rounded,  grayish,  viscid-looking  colonies. 

5.  BACILLUS  TYPHOSUS:  Medium-sized  bacillus  with 
rounded  ends,   generally  short,  but  sometimes   long  or 
thread-like.    Decolorized  by  Gram's  method.    Growth  on 


150  Clinical  Laboratory  Technic 

culture  media  is   similar  but  slower  than  that  of  coli 
communis. 

6.  BACILLUS  TUBERCULOSIS  :  Slender  rods  which  occur 
singly  and  in  pairs,  usually  slightly  curved.    Branching 
and    club-shaped    forms    are    sometimes    observed,    also 
spherical  granules.    Stained  by  Gram's  method.    Stained 
red  by  carbol  fuchsin  stain.    Growth  on  special  media 
appears  as  dry,  cream-colored,  granular  patches. 

7.  BACILLUS  DIPHTHERIA:  These  bacilli  vary  in  size 
and  shape.    They  occur  in  irregular  forms,  often  club- 
like  in  shape,  with  a  constriction  in  the  middle.    They 
show  great  variability  in  the  staining  of  different  parts 
of  the  protoplasm.    Stained  by  Gram's  method.    Loffler's 
and  Neisser's  stains  are  generally  used.    Growth  on  cul- 
ture media  appears  as  round,  elevated,  smooth  colonies. 

Bacteria  of  Pathogenic  Significance  Not  Commonly 
Encountered  in  Pathological  Processes  in  Man 

1.  GONOCOCCI:  Medium-sized  cocci  composed  usually 
of  two  hemispheres  separated  by  a  narrow,  unstained 
interval.     Decolorized  by  Gram's  method,  i.  e.,  they  are 
stained  a  bright  red.    Special  media  is  required,  and  the 
colonies  appear  as  minute,  grayish,  translucent  points. 

2.  AEROGENES  CAPSULATUS  :  Bacilli  varying  in  length, 
ends   rounded   or   square-cut;  occur   singly  or  in  pairs 
and  in  clumps.    Stained  by  Gram's  method.    This  bacillus 
will  not  grow  in  the  presence  of  oxygen.    Colonies  in 
anaerobic  cultures  are  oval,  grayish  to  brownish  white. 

3.  BACILLUS  INFLUENZA  :  Small  bacilli  with  rounded 
ends  of  variable  length.    Sometimes  occur  in  pairs  and 
resemble  pairs  of  cocci.    Growth  on  special  media  appears 
as    small,    glassy,    transparent    points.     Decolorized    by 


Bacteria  151 

Gram's  method.    The  end  may  be  more  deeply  stained 
than  central  portions. 

4.  BACILLUS  ACNE:  Short,  broad  bacillus,  often  show- 
ing beaded  appearance.    Stained  by  Gram's  method.    It 
is  anaerobic ;  small  colonies. 

5.  BACILLUS  ANTHRACIS  :  The  organism  grows  in  long, 
segmented  threads,  varying  in  length.    The  segment  rep- 
resents the  bacillus.    Growth  on  media  appears  as  small, 
white  colonies. 

BACILLUS  PYOCYANEUS  (Bacilli  of  Green  Pus) :  Small 
bacilli  with  rounded  ends.  Decolorized  by  Gram's  method. 
Appearance  on  media  is  slightly  elevated,  viscid  layer  of 
greenish  color. 


Lesions  Caused  by  Bacteria 


Staphylococcus  pyogenes  aureus. 


Streptococcus  pyogenes 


Pneumococcus 

(Diplococcus  Lanceolatus) 


Furuncle, 
Carbuncle, 
Broncho-pneumonia, 
Abscess  of  lung, 
Infection  of  wounds. 

Erysipelas, 

Tonsilitis, 

Septicemia, 

Endocarditis, 

Broncho-pneumonia. 

Broncho-pneumonia, 
Lobar  pneumonia, 
Meningitis, 
Endocarditis, 
Septicemia. 


152  Clinical  Laboratory  Technic 


Salpingitis, 
Vaginitis, 

Peritonitis, 
Cjonococcus    T-   j           •••*• 

_  ,      ,  )   Endocervicitis, 

(Diplococcus  Gonorrhea)  ]   Metritis 

Prostatitis, 
Ophthalmia. 

M.  Catarrhalis Catarrh. 

B.  influenzae Influenza. 

_.  i   Cystitis, 

B.  col.  commums J  Cholecystitis. 


i 


B.  tuberculosis 


Tuber- 
culosis. 


Spine  (Pott's  Disease), 

Lung, 

Skin  (Lupus), 


Lymph-nodes  (Scrofula). 

B.  diphtheriae    Diphtheria. 

Treponema  pallidum Syphilis. 

B.  typhosus   Typhoid  fever. 

{Necrosis  of  wounds, 
Emphysematous  gangrene* 
Gas  cysts. 

B.  anthrax Anthrax. 

B.  pyocyaneus Pus. 

B.  acne    Acne. 

Staining  Methods 

The  glass  surface  of  the  slide  should  be  absolutely 
clean,  and  this  can  be  done  by  burning  the  surface  for  a 
moment  in  a  Bunsen  or  alcohol  flame.  Then  place  a  loop- 
ful  of  distilled  water  on  the  slide,  touch  this  with  the  hot 
platinum  loop,  resterilize  the  loop,  and  transfer  a  colony 
from  the  culture  into  the  loop  of  water.  Stir  with  a 
circular  motion.  Fluid  cultures  do  not  need  dilution. 


Bacteria  153 

When  the  smear  is  dry,  pass  the  slide  three  times  through 
the  flame,  film  side  up,  to  fix  the  preparation. 

The  three  most  important  routine  stains  are  Gram's 
stain,  Loffler's  methylene  blue,  and  carbol  fuchsin  (Ziehl- 
Neelsen). 

Gram's  Stain 

1.  Cover  smear  with  gentian  violet  i  minute. 

2.  Wash  in  tap  water. 

3.  Lugol's  solution  i  minute. 

4.  Wash  in  tap  water. 

5.  Decolorize  with  95%  alcohol  until  no  more  violet 
color  runs  off. 

6.  Wash. 

7.  Counterstain  with  dilute  carbol  fuchsin  or  Bismarck 
brown. 

Smith's  Simplification  of  the  Gram  Stain 

Six  wide-mouthed,  4-ounce  bottles  are  needed  to  hold 
the  following  solutions : 

Bottle  i  contains  alcohol-saturated  gentian  violet,  with 
distilled  water  in  the  proportion  of  i  part  stain  to  3  parts 
water. 

Bottle  2  contains  plain  distilled  water. 

Bottle  3  contains  Gram's  iodine  solution. 

Bottle  4  contains  absolute  methyl  alcohol. 

Bottle  5  contains  plain  distilled  water. 

Bottle  6  contains  Ziehl-Neelsen  carbol  fuchsin. 

METHOD.  Dip  the  slide  into  Bottle  i  from  5  to  10 
seconds,  with  constant  stirring.  Transfer  to  Bottle  2  for 
a  few  seconds,  shake  off  the  excess  of  water,  dip  into 
Bottle  3  for  5  seconds,  with  constant  agitation  of  the  slide. 


154  Clinical  Laboratory  Technic 

Transfer  to  Bottle  4,  where  it  takes  but  a  few  seconds  to 
decolorize,  then  wash  in  Bottle  5  and  lastly  in  Bottle  6  for 
2  seconds.  Wash  in  tap  water. 

The  Gram-positive  bacteria  are  stained  a  deep  violet, 
but  it  must  be  kept  in  mind  that  old,  degenerate,  and  dead 
cultures  do  not  stain  characteristically.  The  pathogenic 
cocci  are  nearly  all  Gram-positive,  with  a  few  exceptions, 
and  the  pathogenic  bacilli  are  Gram-negative,  except  the 
acid-fast  ones,  diphtheria,  and  acne  organisms. 


Gram-Positive  Gram-Negative 

Staphylococcus.  Meningococcus. 

Streptococcus.  M.  melitensis. 

M.  tetragenus.  B.  coli  communis. 

Pneumococcus.  Sp.  cholerae  asiaticse. 

Anthrax  bacillus.  B.  mallei. 

Tubercle  bacillus.  B.  proteus. 

Lepra  bacillus.  B.  of  bubonic  plague. 

Tetanus  bacillus.  B.  of  Koch-Weeks. 

Diphtheria.  M.  catarrhalis. 

B.  aerogenes  capsulatus.  B.  typhosus. 

Oi'dium  albicans.  B.  dysenterise  (Shiga). 

Mycelium  of  actinomyces.  B.  pyocyaneus. 

Saccharomyces.  B.  pneumonia   (Friedlander), 

Hoffmann's  bacillus.  B.  of  influenza. 

B.  xerosis.  B.  of  chancroid. 

B.  acne.  Gonococcus. 


LofHer's  Stain  for  Diphtheria 

1.  Cover  smear  with  Loffler's  alkaline  methylene  blue 
for  2  minutes. 

2.  Wash  in  tap  water ;  dry. 

Note.  Bacilli  which  resemble  B.  diphtheria.  Diphtheroid 
bacilli  rarely  give  the  blue  dot  staining  at  the  two  ends.  They 
usually  stain  solidly.  They  are  shorter,  thicker,  and  do  not  curve 
as  gracefully.  They  are  nonpathogenic  for  guinea-pigs.  Growth 
is  luxuriant.  Xerosis  bacillus  also  resembles  the  diphtheria 
bacillus.  It  is  nonyirulent  for  guinea-pigs.  Does  not  show 
Neisser's  granule  stain.  Produces  very  little  acid  in  sugar  media. 


/a 


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— After  Gibson 

I,  Staphylococci ;  2,  streptococci ;  3,  M.  tetragenus ;  4,  pneu- 
mococci ;  5,  anthrax ;  6,  B.  tuberculosis ;  7,  B.  leprae ;  8,  B.  smeg- 
matis;  9,  B.  tetanus;  10,  B.  diphtherise ;  n,  B.  Hoffmann! 
(diphtheroids)  ;  12,  B.  aerogenes  capsulatus;  13,  B.  xerosis;  14, 
actinomyces,  clubs,  filaments;  15,  oidium  albicans ;  16,  meningo- 
cocci;  17,  B.  Koch-Weeks;  18,  Sp.  cholerae  asiaticse;  19,  B.  mallei 
(Glanders);  20,  B.  proteus;  21,  B.  of  bubonic  plague;  22,' M. 
catarrhalis;  23,  B.  typhosus;  24,  B.  dysenteriae  (Shiga)  ;  25,  B. 
pneumonia  (Friedlander)  ;  26,  B.  of  influenza;  27,  gonococci; 
28,  B.  pyocaneus;  29,  B.  coli  communis;  30,  B.  of  Ducrey 
(chancroid). 

155 


156  Clinical  Laboratory  Technic 

Neisser's  Method  (for  Diphtheria) 

This  method  accentuates  the  deeply  stained  granules 
in  the  bodies  of  the  individual  bacilli. 

1.  Cover  with  Neisser's  solution  No.  I  for  5  seconds. 

2.  Wash  in  tap  water. 

3.  Bismarck  brown,  3  to  5  seconds. 

4.  Wash  in  tap  water ;  dry. 

Ziehl-Neelsen  Stain  for  Tubercle  Bacilli 

1.  Cover  smear  with  carbol  fuchsin,  steaming  I  to  5 
minutes. 

2.  Sulphuric  acid  (20%),  20  seconds. 

3.  Alcohol  (95%)  until  no  more  color  will  come  out. 

4.  Wash  in  tap  water. 

5.  Cover  with  Loffler's  methylene  blue  10  seconds. 

6.  Wash  in  tap  water ;  dry. 

Gabbet's  Method. — Tubercle  Bacilli 

1.  Carbol  fuchsin,  steaming  i  to  5  minutes. 

2.  Wash  in  tap  water. 

3.  Cover  with  Gabbet's  solution  i  minute. 

4.  Wash  in  water ;  dry. 

The  tubercle  bacillus  may  be  confounded  with  the 
bacillus  of  leprosy  and  the  smegma  bacillus.  It  may  be 
differentiated  from  the  smegma  bacillus  by  the  fact  that 
it  is  not  decolorized  by  alcohol  after  treatment  with  acid. 

The  bacillus  of  leprosy  stains  more  easily  and  loses  its 
color  more  quickly  than  the  tubercle  bacillus.  They  occur 
in  greater  numbers  and  are  frequently  beady.  They  do 
not  produce  lesions  when  injected  into  guinea  pigs. 


Bacteria 


157 


Moeller's  Spore  Stain 

1.  Chloroform  (in  covered  dish),  2  minutes. 

2.  Chromic  acid  (5%),  I  minute. 

3.  Wash  thoroughly  in  water. 

4.  Carbol  fuchsin,  I  to  3  minutes'  steaming. 

5.  Decolorize  with  sulphuric  acid  (i%)   until  only  a 
light  pink  color  remains. 

6.  Wash  in  water. 

7.  Saturated  aqueous  methylene  blue,  i  minute. 

8.  Wash,  dry,  and  mount. 

The  bodies  of  the  bacteria  are  stained  by  the  methylene 
blue,  and  the  spores  are  stained  by  the  fuchsin  and  appear 
red. 

Welch's  Method  for  Capsules 

1.  Cover  with  glacial  acetic  acid  for  a  few  seconds. 

2.  Drain  with  filter  paper  and  place  in  a  dish  of  aniline 
water  gentian  violet  for  a  few  seconds. 

3.  Wash  in  solution  of  sodium  chloride  (2%). 

4.  Examine  in  this  same  solution. 

Bacteria  stain  a  dark  violet,  and  the  capsules  a  pale 

violet. 

\ 

Loffler's  Method  for  Staining  Flagella 

i.  Cover  with  freshly  filtered  mordant  solution  con- 
sisting of  : 

Aqueous  solution  of  tannic  acid  (20  grams 

tannic  acid  to  100  c.c.  water)  10  c.c. 

Cold    saturated    solution   of    ferrous    sul- 
phate 5  c.c. 
Saturated  aqueous  or  alcoholic  solution  of 

gentian  violet  or  fuchsin  I  c.c. 


158  Clinical  Laboratory  Technic 

2.  Heat  gently  for  i  minute. 

3.  Wash  in  water. 

4.  Cover  with  freshly  prepared  and  filtered  solution 
of  aniline  gentian  violet  or  aniline  fuchsin,  and  heat  gently 
for  i  minute. 

5.  Wash  in  water. 

Syphilis 

Cleanse  the  lesions  from  any  adherent  exudate,  and 
make  a  smear  preparation  from  the  juice  of  the  tissue 
obtained  by  pressure  and  scraping.  Dry  and  stain.  The 
smear  may  be  fixed  and  stained  as  a  blood  smear  with 
Wright's  stain. 

A  B 


— After  Gibson 
SPIROCH^ETA  PALLIDA 

A.  Giemsa's  Stain.    The  spirochetes  and  bacteria  are  stained 
purple. 

B.  India-ink.     The  spirochetes  and  bacteria  appear  as  un- 
stained bodies  in  a  black  background. 

Giemsa's  Method 

1.  Fix  the  smear  by  drawing  three  times  through  the 
flame,  or  placing  iri  absolute  alcohol  15  minutes. 

2.  Dilute  10  drops  of  Giemsa's  stain  with  10  C.C.  of 


Bacteria  159 

distilled  water  and  cover  the  smear.  This  process  is  re- 
peated four  or  five  times  at  15 -second  intervals.  The 
parasites  take  an  intense  dark  red  stain. 

Another  Method. — Mallory  and  Wright 

1.  After  fixing  smear  with  heat,  cover  with   10  c.c. 
of  a  5%   solution  of  glycerin  mixed  with   13  drops  of 
Giemsa's  stain,  which  has  been  heated  to  60°  C. 

2.  Allow  this  hot  solution  to   remain   on  the   smear 
15  to  30  minutes.    Wash  in  water. 

If  intense  staining  is  desired,  add  to  the  distilled  water 
before  mixing  it  with  the  stain  i  or  2  drops  of  i%  potas- 
sium carbonate  solution  to  10  c.c.  of  water. 

India  Ink  Stain 

Place  5  loopfuls  of  Chin-Chin  India  Ink  near  the  end 
of  a  slide ;  add  5  loopfuls  of  distilled  water  and  i  loop- 
ful  of  material  to  be  examined.  Mix,  then  place  the  top 
of  another  slide  on  half  of  the  first  slide,  press  together, 
and  pull  apart  sideways.  Dry  and  examine. 

Label  all  cultures  and  smears  with  the  patient's  name, 
date,  source,  and  hospital  number. 


Bacteria 


Name   

Date.. 

Source 

Remarks.. 


160  Clinical  Laboratory  Technic 

Distribution  of  Bacteria  in  the  Animal  Body1 

SKIN.  Staphylococci  and  streptococci.  Tubercle,  leprosy, 
smegma,  tetanus,  gas,  and  anthrax  bacilli. 

NOSE  AND  THROAT.  Staphylococci,  streptococci,  pneumo- 
cocci,  diphtheria,  influenza,  and  pertussis  organisms. 
Meningococci,  M.  catarrhalis,  tubercle  bacillus,  and 
virus  of  poliomyelitis. 

EAR  AND  EYE.  Staphylococcus,  streptococcus,  pneumo- 
coccus,  gonococcus,  diphtheria,  influenza,  Koch- 
Weeks  and  Morax-Axenfeld  bacilli. 

LUNGS.  Staphylococcus,  streptococcus,  pneumococcus', 
tubercle,  Friedlander's,  influenza,  pertussis,  colon- 
typhoid,  anthrax  and  plague  bacilli. 

PELVIC  ORGANS.  Staphylococcus,  streptococcus,  gono- 
coccus, tubercle  and  smegma  bacilli,  and  Spirochaeta 
pallida. 

SEROUS  FLUIDS,  i.  Cerebrospinal  fluid  :  (a)  clear  fluid: 
tubercle  bacillus,  Spirochaeta  pallida,  virus  of  polio- 
myelitis; (b)  turbid  fluid:  pneumococcus,  strepto- 
coccus, meningococcus,  B.  influenza,  typhoid-colon 
group.  2.  Pleural  and  pericardial  fluids :  (a)  clear; 
tubercle  bacillus;  (b)  turbid  fluid:  pneumococcus, 
streptococcus,  B.  influenza,  typhoid,  Staphylococcus. 
3.  Peritoneal  fluid:  Streptococcus  group;  colon- 
typhoid  group ;  tubercle  bacillus. 

BLOOD.  Streptococcus  and  pneumococcus,  typhoid,  Staphy- 
lococcus groups.  Recurrent  fever  and  Spirochaeta. 
Plague  bacilli.  Friedlander's  bacillus  (rare). 

INTESTINAL  CONTENTS  AND  FECES.  Colon-typhoid  group, 
paratyphoids,  dysentery  and  B.   fecalis  alkaligenes. 
Mucosus    capsulatus    group.      Tubercle   bacilli,   an- 
thrax, tetanus  and  gas  bacilli. 
1  Classification  by  Kendall. 


Bacteria  161 


Descriptive  Chart.    Society  of  American 
Bacteriologists 

Glossary  of  Terms 

AGAR  HANGING  BLOCK:  A  small  block  of  nutrient  agar 
cut  from  a  poured  plate  and  placed  on  a  cover  glass, 
the  surface  next  the  glass  having  been  first  touched 
with  a  loop  from  a  young  fluid  culture  or  with  a 
dilution  from  the  same.  It  is  examined  upside  down, 
the  same  as  a  hanging  drop. 

AMEBOID:  Assuming  various  shapes  like  an  ameba. 

AMORPHOUS  :  Without  visible  differentiation  in  structure. 

ARBORESCENT  :  A  branched,  tree-like  growth. 

BEADED  :  Stab  or  stroke,  disjointed  or  semi-confluent 
colonies  along  the  line  of  inoculation. 

BRIEF  :  A  few  days,  a  week. 

BRITTLE  :  Growth  dry,  friable  under  platinum  needle. 

BULLATE:  Growth  rising  in  convex  prominences,  like  a 
blistered  surface. 

BUTYROUS  :  Growth  of  a  butter-like  consistency. 

CHAINS:  Short  chains,  composed  of  2  to  8  elements. 
Long  chains,  composed  of  more  than  8  elements. 

CILIATE  :  Having  fine,  hair-like  extensions  like  cilia. 

CLOUDY:  Fluid  cultures  which  do  not  contain  pseudo- 
zoogloese. 

COAGULATION  :  The  separation  of  casein  from  whey  in 
milk.  This  may  take  place  quickly  or  slowly,  and 
as  the  result  either  of  the  formation  of  an  acid  or  of 
a  lab  ferment. 

CONTOURED:  An  irregular,  smoothly  undulating  surface, 
like  that  of  a  relief  map. 


1 62  Clinical  Laboratory  Technic 

CONVEX  :  Surface  the  segment  of  a  circle,  but  flattened. 

COPROPHYL  :  Dung  bacteria. 

CORIACEOUS  :  Growth  tough,  leathery,  not  yielding  to  the 

platinum  needle. 
CRATERIFORM  :  Round,  depressed,  due  to  the  liquefaction 

of  the  medium. 

CRETACEOUS  :  Growth  opaque  and  white,  chalky. 
CURLED:  Composed  of  parallel  chains  in  wavy  strands, 

as  in  anthrax  colonies. 
DIASTASIC  ACTION:   Same  as  Diastatic;  conversion   of 

starch  into  water-soluble  substances  by  diastase. 
ECHINULATE:  In  agar  stroke,  a  growth  along  line  of  in- 
oculation, with  toothed  or  pointed  margins ;  in  stab 

cultures,  growth  beset  with  pointed  outgrowths. 
EFFUSE  :  Growth  thin,  veily,  unusually  spreading. 
ENTIRE:  Smooth,  having  a  margin  destitute  of  teeth  or 

notches. 

EROSE  :  Border  irregularly  toothed. 
FILAMENTOUS:   Growth   composed   of   long,   irregularly 

placed,  or  interwoven  filaments. 
FILIFORM  :  In  stroke  or  stab  cultures,  a  uniform  growth 

along  line  of  inoculation. 
FIMBRIATE  :  Border  fringed  with  slender  processes,  larger 

than  filaments. 
FLOCCOSE:   Growth  composed  of   short,  curved  chains, 

variously  oriented. 
FLOCCULENT  :  Fluids  which  contain  pseudozoogloese,  i.  e.y 

small,  adherent  masses  of  bacteria  of  various  shapes, 

floating  in  the  culture  fluid. 
GRAM'S  STAIN  :  A  method  of  differential  bleaching  after 

gentian  violet,  etc. 
GRUMOSE:  Clotted. 


Bacteria  163 

INFUNDIBULIFORM  :  Form  of  a  funnel  or  inverted  cone. 

IRIDESCENT:  Like  mother-of-pearl.  The  effect  of  very 
thin  films. 

LACERATE:  Having  the  margin  cut  into  irregular  seg- 
ments, as  if  torn. 

LOBATE:  Border  deeply  undulate,  producing  lobes. 

LONG:  Many  weeks  or  months. 

MAXIMUM  TEMPERATURE:  Temperature  above  which 
growth  does  not  take  place. 

MEDIUM  :  Several  weeks. 

MEMBRANOUS:  Growth  thin,  coherent,  like  a  membrane. 

MINIMUM  TEMPERATURE:  Temperature  below  which 
growth  does  not  take  place. 

MYCELIOID:  Colonies  having  the  radiately  filamentous 
appearance  of  mold  colonies. 

NAPIFORM  :  Liquefaction  with  the  form  of  a  turnip. 

NITROGEN  REQUIREMENTS  :  The  necessary  nitrogenous 
food.  This  is  determined  by  adding  to  nitrogen- 
free  media  the  nitrogen  compound  to  be  tested. 

OPALESCENT  :  Resembling  the  color  of  an  opal. 

OPTIMUM  TEMPERATURE:  Temperature  at  which  growth 
is  most  rapid. 

PELLICLE  :  In  fluid  bacterial  growth,  either  forming  a  con- 
tinuous or  an  interrupted  sheet  over  the  fluid. 

PEPTONIZED  :  Curds  dissolved  by  trypsin. 

PERSISTENT:  Many  weeks  or  months. 

PLUMOSE:  A  fleecy  or  feathery  growth. 

PSEUDOZOOGLCE^  :  Clumps  of  bacteria,  not  dissolving 
readily  in  water,  arising  from  imperfect  separation 
or  more  or  less  fusion  of  the  components,  but  not 
having  the  degree  of  compactness  and  gelatinization 
seen  in  zoogloese. 


164  Clinical  Laboratory  Technic 

PULVINATE:  The  form  of  a  cushion,  decidedly  convex. 

PUNCTIFORM:  Very  minute  colonies,  at  the  limit  of 
natural  vision. 

RAISED:  Growth  thick,  with  abrupt  or  terraced  edges. 

RAPID  :  Developing  in  24  to  48  hours. 

REPAND  :  Wrinkled. 

RHIZOID  :  Growth  of  an  irregular  branched  or  root-like 
character,  as  in  B.  mycoides. 

RING:  Same  as  Rim;  growth  at  the  upper  margin  of  a 
liquid  culture,  adhering  more  or  less  closely  to  the 
glass. 

SACCATE  :  Liquefaction  the  shape  of  an  elongated  sack, 
tubular,  cylindrical. 

SCUM  :  Floating  islands  of  bacteria,  an  interrupted  pelli- 
cle, or  bacterial  membrane. 

SLOW  :  Requiring  5  or  6  days  or  more  for  development. 

SPORANGIA:  Cells  containing  endospores. 

SPREADING:  Growth  extending  beyond  the  line  of  inocu- 
lation. 

STRATIFORM  :  Liquefying  to  the  walls  of  the  tube  at  the 
top  and  then  proceeding  downward  horizontally. 

THERMAL  DEATH-POINT:  The  degree  of  heat  required 
to  kill  young  fluid  cultures  of  an  organism  exposed 
for  10  minutes  (in  thin-walled  test  tubes  of  a  diam- 
eter not  exceeding  20  mm.)  in  a  thermal  water  bath. 
The  water  must  be  kept  agitated  so  that  the  temper- 
ature shall  be  uniform  during  the  exposure. 

TRANSIENT  :  A  few  days. 

TURBID:  Cloudy,  with  flocculent  particles;  cloudy  plus 
flocculence. 

UMBONATE:  Having  a  button-like,  raised  center. 

UNDULATE  :  Border  wavy,  with  shallow  sinuses. 


Bacteria  165 

VERRUCOSE:  Growth  wart-like,  with  wart-like  promi- 
nences. 

VERMIFORM-CONTOURED:  Growth  like  a  mass  of  worms 
or  intestinal  coils. 

VILLOUS  :  Growth  beset  with  hair-like  extensions. 

VISCID:  Growth  follows  the  needle  when  touched  and 

V 

withdrawn;  sediment  on  shaking  rises  as  a  coherent 
swirl. 

ZOOGLCE^E:  Firm,  gelatinous  masses  of  bacteria,  one  of 
the  most  typical  examples  of  which  is  the  Strepto- 
coccus mesenterioides  of  sugar  vats  (Leuconostoc 
mesenterioides) ,  the  bacterial  chains  being  sur- 
rounded by  an  enormously  thickened,  firm  covering, 
inside  of  which  there  may  be  one  or  many  groups 
of  the  bacteria. 


CHAPTER  IX 
CULTURE  MEDIA 

Culture  media  consist  of  various  liquid  and  solid  nutri- 
tive substances  in  or  upon  which  bacteria  will  grow,  and 
the  media  must  be  adjusted  to  the  peculiarities  of  the 
individual  bacteria. 

Sterile  test  tubes  are  used  as  containers,  stoppered  with 
cotton  which  has  been  dipped  in  melted  paraffin,  then 
inserted  into  the  tube.  This  prevents  evaporation  and 
the  invasion  of  molds. 

The  most  common  media  have  for  their  basis  an  extract 
or  infusion  of  meat,  to  which  peptone  and  sodium  chloride 
are  added. 

If  fresh  meat  is  used,  take  500  grams  (i  pound)  of 
lean  meat,  finely  chopped.  Cover  with  1,000  c.c.  of  water.1 
Place  in  the  ice  chest  over  night.  The  following  day 
skim,  strain,  and  make  the  infusion  up  to  1,000  c.c.  Then 
add  10  grams  of  peptone  and  5  grams  of  sodium  chloride 
which  have  been  dissolved  in  a  few  c.c.  of  the  infusion. 

Boil  10  minutes,  then  place  on  the  scales  and  balance 
with  its  counterpoise  and  I  kilo  weight  on  the  other  side. 
Add  sufficient  hot  water  to  the  media  to  make  up  to 
1,000  c.c.  Boil  5  minutes;  test  for  acidity. 

The  reaction  may  be  tested  with  litmus  paper  and  dilute 
HC1  added  when  the  media  becomes  too  alkaline.  This 

'The  meat  may  be  boiled  for  1  hour;  filter  and  make  up  to 
1,000  c.c. 

1 66 


Culture  Media  167 

method  is  sufficient  for  the  cultivation  of  bacteria  for 
ordinary  purposes. 

Bacteria  are  susceptible  to  slight  variations  in  the  acidity 
and  alkalinity  of  media,  and  the  reaction  has  a  marked 
effect  upon  their  morphology  and  mode  of  growth.  In 
the  cultivation  of  bacteria  of  the  air,  soil,  and  water,  a 
more  exact  adjustment  of  the  reaction  is  made  by  titrating, 
using  phenolphthalein  as  an  indicator. 

The  neutral  point  of  litmus  corresponds  with  a  reaction 
of  +1.5  to  phenolphthalein. 

Titration 

1  Media  in  the  process  of  preparation  is  usually  acid; 

N 
sometimes  5  c.c.  of  -     HC1  are  added. 

N 
Fill  the  burette  with  —  NaOH,  make  a  reading,  and 

20 

record. 

5  c.c.  of  the  medium  to  be  tested  are  measured  in  a 
pipette  and  transferred  into  a  small  porcelain  dish.  Add 
45  c.c.  of  hot  water  (cold  water  contains  carbon  dioxide 
and  might  give  alkalinity  to  the  media)  and  5  drops  of 
phenolphthalein  (0.5%  in  50%  alcohol). 

Place  this  small  dish  under  the  burette  and  carefully 

run  in  the  —  NaOH  until  a  distinct  pink  color  remains 

20  t:  •*". 

in  the  fluid  after  stirring. 

aTo  make  more  acid:  Add  to  each  100  c.c.  of  the  medium  i  c.c. 
of  normal  HC1  for  each  degree  that  the  reaction  is  to  be  changed. 
EXAMPLE,  i  liter  of  medium  with  a  reaction  of  .8  degree  is  to  be 
made  i ;  then  0.2  c.c.  of  normal  HC1  should  be  added  to  each 
100  c.c.,  or  2  c.c.  for  the  liter.  To  make  more  alkaline,  add  normal 
NaOH. 


1 68  Clinical  Laboratory  Technic 

EXAMPLE.  0.4%  acidity  is  desired  (standard  required 
by  the  American  Public  Health  Association;  i.e.,  1.5% 
normal  NaOH  added  to  medium  makes  it  neutral  to 
phenolphthalein).  Adjustment  of  the  reaction  to  this 
standard  is  made  by  adding  to  the  medium 

-  NaOH  or  -  HC1 
i  i 

The  first  reading  of  the  burette  is  25,  and  after  adding 
to  the  medium  and  obtaining  the  pink  color,  the  burette 
reads  25.8.  The  titration  shows  that  5  c.c.  of  the  medium 

N 

requires  0.8  c.c.  —  NaOH  to  make  it  neutral  to  phenol- 
phthalein, therefore  the  total  amount  will  require  0.8%, 
or  8  c.c.,  for  a  liter.  25.8  —  25  =  0.8.  The  reaction  re- 

N 
quired  is  such  that  0.4%   of  the  —  NaOH  should  be 

used  to  make  the  medium  neutral.  Subtracting  0.4  from 
0.8,  we  have  as  a  result  0.4  excess  acidity.  Therefore 
to  every  5  c.c.  in  the  1,000  c.c.  of  medium,  0.4  c.c.  of 

N 

the  —  NaOH  is  added  to  neutralize  the  acidity,  or  the 
20 

N  N 

-  NaOH  may  be  used.    1,000  -f-  5  X  0.4  =  80  c.c.  — • 

NaOH,  or  4  c.c.  ^  NaOH. 

When  the  titration  is  finished,  cool  the  medium  to 
60°  C. ;  add  3  eggs  which  have  been  beaten  lightly  with 
200  c.c.  of  cold  water.  Boil  20  minutes,  stirring  occa- 
sionally. Filter,  and  run  10  c.c.  in  sterile  test  tubes. 
Sterilize  |  hour  at  120°  C.  on  3  successive  days. 


Culture  Media  169 

Bouillon 

Formula  for  1,000  c.c. : 

Beef  extract  (Liebig's)  3  grams 

Or  lean  beef  500       " 

Peptone  (Witte's)  10      " 

Sodium  chloride  5       " 

Water  1,000  c.c. 

Glucose  Bouillon 

Add   10  grams  of  glucose  to  the  preceding  bouillon 
medium. 

Agar-agar  (plain) 
Formula  for  1,000  c.c.: 

Agar-agar  15  grams 

Beef  extract  3       " 

Or  lean  beef  500      " 

Peptone  10      " 

Sodium  chloride  5       " 

Water  1,000  c.c. 

Filter  into  a  1,000  c.c.  flask,  sterilize  \  hour,  filter  again 

to  remove  the  precipitate  of  phosphates,  and  run  10  c.c. 

into  sterile  test  tubes.    Slant  tubes  while  cooling  after  the 

third  sterilization. 

Glucose  Agar-agar 

Add  10  grams  of  glucose  to  the  agar-agar  medium. 

Glycerin  Agar-agar 

Add  60  c.c.  of  glycerin  to  the  plain  agar-agar  medium 
after  its  filtration. 


170  Clinical  Laboratory  Technic 

Gelatin  (plain) 

Formula  for  1,000  c.c. : 

Dissolve  100  grams  of  golden  seal  French  gelatin  in 
1,000  c.c.  of  bouillon.  Boil  5  minutes.  Neutralize  the 
acidity  of  the  gelatin  with  a  10%  solution  of  caustic  soda 
to  a  faint  alkalinity.  Cool  to  60°  C.  and  beat  an  egg  into 
it,  then  boil  10  minutes.  Filter  and  run  into  test  tubes, 
10  c.c.  for  "slant"  and  15  c.c.  for  "stab"  cultures. 

Fill  the  chamber  of  the  sterilizer  with  steam  before 
putting  in  the  media.  Sterilize  15  minutes,  3  successive 
days,  at  10  pounds  pressure.  Plunge  tubes  in  cold  water 
after  each  sterilization. 

Sugar-free  Bouillon. — Method  No.  1 

Formula  for  1,000  c.c.: 

Chopped  lean  beef  500  grams 

Water  1,000  c.c. 

Place  in  the  ice  chest  12  to  24  hours.  Filter,  and  add 
a  24-hour  bouillon  culture  of  colon  bacillus.  Neutralize, 
then  incubate  12  hours.  Add  the  10  grams  of  peptone, 
5  grams  of  sodium  chloride,  and  heat  gently  until  the 
peptone  dissolves,  then  boil  30  minutes.  Balance  and  add 
water  to  make  up  for  loss  by  evaporation.  Titrate,  filter, 
and  store  in  tubes  or  flasks.  Sterilize. 

Blood  Serum. — Lbffler's 
Formula : 

Glucose  bouillon  I  part  (300  c.c.) 

Beef  blood  serum  3  parts   (900  c.c.) 

The  blood  serum  is  collected  at  the  abattoir  in  thor- 
oughly clean  glass  jars.  Place  in  a  cool  place  24  to  48 


Culture  Media  171 

hours,  to  allow  it  to  clot  and  the  serum  to  separate. 
Remove  the  serum  with  a  sterile  pipette. 

Slant  tubes  and  solidify  by  heating  3  hours  at  80°  to 
90°  C. 

Sterilize  in  Arnold  sterilizer  20  to  30  minutes  on 
3  successive  days. 

Dextrose  bouillon  may  be  used  instead  of  glucose,  and 
this  is  made  by  adding  \%  dextrose  to  the  plain  bouillon. 
B.  diphtheria  is  cultivated  on  this  media. 

Litmus  Milk 

Formula : 

Fresh  milk  .    300  c.c. 

Litmus  solution  50  c.c. 

Take  fresh  milk  and  place  in  the  autoclave  for  i  hour 
at  60°  C.,  then  skim.  Boil  8  litmus  cubes  in  100  c.c.  of 
water,  filter,  and  add  sufficient  to  give  the  milk  a  pale  blue 
color.  Sterilize  30  minutes  at  4  or  5  pounds  pressure  on 
3  successive  days. 

This  media-  is  used  for  determining  certain  of  the 
physiological  properties  of  bacteria — whether  they  pro- 
duce in  their  growth  acid  or  alkali.  Colon  bacilli  turn 
litmus  milk  pink.  Typhoid  and  colon  bacilli  are  some- 
times confused;  they  can  be  differentiated  with  litmus 
milk. 

Egg  Medium. — Dorset 

Formula : 

Fresh  eggs  4 

Sterile  distilled  water  25  c.c. 

Fresh  eggs  are  broken  under  aseptic  conditions  into  a 
sterile  graduate.  To  every.  4  eggs  add  25  c.c.  of  sterile 


172  Clinical  Laboratory  Technic 

distilled  water,  mixing  the  whites  and  yolks  thoroughly 
with  a  sterile  glass  rod.  Strain  the  mixture  through  a 
sterile  cloth,  then  run  10  c.c.  into  sterile  tubes,  and  slowly 
harden  in  form  of  "slants"  in  the  autoclave  4  hours  at 
73°  to  76°  C.  on  3  successive  days.  This  medium  is 
used  for  the  cultivation  of  tubercle  bacilli.  Just  before 
inoculating  the  medium,  add  I  c.c.  of  sterile  distilled 
water  to  each  tube,  to  supply  the  moisture  required  for 
the  development  of  the  bacilli,  and  incubate  over  night  at 
38°  C.  Colonies  should  become  visible  after  7  days. 

Modification  of  Endo's  Medium 

Formula : 

Cold  water  1,000  c.c. 

Powdered  agar  15  grams 

Peptone  (Witte's)  10 

Beef  extract  (Liebig's)  3 

Cook  i  hour  in  a  double  boiler,  then  add  enough  of  a 
10%  solution  of  sodium  carbonate  to  make  slightly  alka- 
line to  litmus.  Filter,  and  sterilize  i  hour  at  15  pounds 
pressure.  To  i  c.c.  of  10%  solution  of  fuchsin  (made 
up  with  96%  alcohol)  add  10  c.c.  of  freshly  prepared 
10%  solution  of  sodium  sulphite  (anhydrous).  Arnoldize 
20  minutes,  then  add  i%  of  this  solution  and  i%  of 
lactose  (C.  P.)  to  the  agar  medium.  Sterilize  30  m'.nutes 
at  15  pounds. 

When  used,  the  medium  is  melted  and  poured  into 
sterile  Petri  dishes,  and  left  uncovered  until  the  agar  is 
solid ;  then  inoculate  with  a  suspension  of  feces  in  sugar- 
free  broth.  Incubate  i  hour. 

Rub  the  infected  sugar-free  broth  over  the  surface  of 
the  Petri  dish  with  a  sterile  glass  rod.  Incubate  18  hours. 


Culture  Media  173 

B.  coli  neutralize  the  action  of  the  sodium  sulphite  by 
forming  acid  from  the  lactose,  and  the  colonies  are  red. 
Typhoid,  dysentery,  and  paratyphoid  bacilli  remain  color- 
less and  do  not  form  colonies. 

Jackson's  Lactose  Bile  Media 

To  10  c.c.  of  ox  bile  from  the  abattoir,  or  from  human 
bile  from  cases  of  gall  bladder  drainage  which  has  been 
sterilized,  add  i%  of  peptone  and  i%  lactose.  Tube  in 
fermentation  tubes. 

B.  typhosus  and  B.  coli  will  outgrow  all  other  micro- 
organisms on  this  media.  Arnoldize  ^  hour  on  3  suc- 
cessive days  at  73°  to  76°. 

Special  Media  for  Gonococcus 

No.  i  No.  2 

Agar-agar  (2%)      2  parts      Agar-agar  2  parts 

Hydrocele  fluid         I  part        Blood  (human)         i  part 

Melt  the  agar-agar  and  bring  to  a  temperature  of 
40°  C.  in  a  water  bath.  Add  the  sterile  hydrocele  fluid 
or  blood. 

The  tubes  may  then  be  inoculated  and  their  contents 
poured  into  sterilized  Petri  dishes. 

A  Substitute  for  Ordinary  Blood  Serum. — Stitt's 
Glucose  bouillon  (i%)  10  to  15  c.c. 

Eggs  i 

Make  a  smooth  mixture  in  a  mortar  and  tube.  Inspis- 
sate and  sterilize  as  for  ordinary  serum  slants. 


1/4  Clinical  Laboratory  Technic 

Gibson's  Modification 

Formula  for  500  c.c. : 

Glucose  bouillon  (i%)  120  c.c. 

Eggs  8 

Glycerin  20  c.c. 

Wash  eggs  with  water,  then  with  70%  alcohol.  The 
eggs  are  then  broken  under  aseptic  precautions  into  a 
wide-mouthed  receptacle  (sterile),  and  mixed  thoroughly 
with  the  glucose  bouillon  and  glycerin.  Strain  through  a 
sterile  cloth  and  run  10  c.c.  of  the  mixture  into  sterile 
test  tubes,  and  slowly  harden  in  the  form  of  "slants"  in 
the  autoclave  at  3  or  4  pounds  pressure  for  2  hours  on 
3  successive  days.  The  door  of  the  autoclave  should 
be  left  partly  open. 

Sugar-free  Bouillon. — Method  No.  2 

1.  Bouillon,  1,000  c.c. 

2.  Cool  in  a  flask  and  add  10  c.c.  of  a  24-hour  culture 
of  B.  coli  communis. 

3.  Stopper  with  cotton;  incubate  18  hours.    (The  bac- 
teria ferment  and  destroy  any  sugar  present,  and  render 
the  broth  sugar-free  and  acid.) 

4.  Heat  thoroughly,  to  kill  the  colon  bacilli. 

5.  Place  15  grams  of  purified  talcum  (U.  S.  P.)  in  a 
mortar;    add   the    bouillon    culture,    stirring   constantly. 
Filter  until  the  bouillon  is  perfectly  clear. 

Sugar  Bouillon 

In  special  work  the  following  formula  is  used : 
Sugar-free  bouillon  1,000  c.c. 

Glucose,  or  other  pure  sugars  10  c.c. 


Culture  Media  175 

Dissolve  and  tube  in  Durham's  or  ordinary  fermenta- 
tion tubes. 

Sterilize  several  times  at  5  pounds  pressure,  never 
heating  over  15  minutes  at  a  time,  as  heat  changes  the 
sugars. 

The  amount  of  sugar  in  bouillon  made  from  Liebig's 
meat  extract  is  so  small  that  for  ordinary  purposes  the 
sugar  is  added  directly  to  the  bouillon. 

Special  Media  for  Ameba. — Musgrave  and  Clegg 
Agar-agar  20  grams 

Sodium  chloride  3  grams 

Beef  extract  3  grams 

Water  1,000  c.c. 

The  agar  should  be  i%  alkaline  to  phenolphthalein. 
Place  the  material  containing  the  ameba  in  a  sterile 
flask ;  add  i  c.c.  of  alkaline  bouillon  to  each  100  c.c.  of 
material.  Set  aside  24  to  48  hours.  Inoculate  Petri 
plates  containing  the  nutrient  agar  with  a  loopful  of 
material  from  the  surface  of  the  flask.  Incubate  at  37°  C. 

Differential  Food  Media. — Hiss.    (Mt.  Prospect 
Laboratory,  Brooklyn,  N.  Y.) 
Agar  15  grams 

Gelatin  15       " 

Liebig's  extract  5       " 

Sodium  chloride  5       " 

Dextrose  10      " 

Distilled  water  1,000  c.c. 

Reaction,  1.0%  normal  acid. 

This  medium  is  semi-fluid  at  37°  C.,  and  B.  typhosus 


176  Clinical  Laboratory  Technic 

has  a  tendency  to  swim  out  from  the  colonies,  forming 
branch-like  processes  which  distinguish  it  from  B.  coli. 

Hesse's  Media  for  Typhoid.     (Mt.  Prospect  Labora- 
tory, Brooklyn,  N.  Y.) 

Agar  5  grams 

(4.5  grams  absolutely  dry) 
Peptone  (Witte)  10  grams 

Liebig's  extract  of  beef  5       " 

Salt  8.5       " 

Distilled  water  1,000  c.c. 

4.5  grams  of  agar  are  dissolved  in  500  c.c.  of  distilled 
water,  making  up  the  loss  in  weight  by  evaporation. 
Dissolve  10  grams  of  peptone,  5  grams  of  meat  extract, 
and  8.5  grams  of  salt  in  500  c.c.  of  distilled  water;  the 
loss  in  weight  by  evaporation  is  made  up  by  adding 
distilled  water.  Add  the  two  solutions  together,  boil 
30  minutes,  make  up  loss  in  weight,  then  filter  through 
absorbent  cotton  held  in  the  funnel  by  cotton  flannel. 
Filter  until  perfectly  clear.  Test  the  reaction  and  adjust 
to  1.0%  normal  acid.  Sterilize  20  minutes  at  15  pounds 
pressure.  Cool  with  running  tap  water  and  store  in  ice 
chest. 

In  cases  of  infected  water  and  milk  the  preliminary 
cultivation  in  bile  is  necessary,  in  order  to  increase  the 
typhoid  germs  in  numbers  over  the  various  other  species 
always  present  in  contaminated  supplies. 

METHOD.  Place  8  tubes  containing  9  c.c.  of  sterile 
distilled  water  in  a  rack,  together  with  8  sterile  Petri 
dishes.  Number  the  tubes  and  plates  from  I  to  8. 

Into  tube  No.  i  place  i  c.c.  of  feces,  water,  or  milk  to 
be  tested,  and  which  has  been  previously  inoculated  and 


Culture  Media  177 

incubated  at  least  24  hours.  Mix  thoroughly  with  9  c.c. 
of  distilled  water,  then  place  i  c.c.  of  this  mixture  in  plate 
No.  i  and  i  c.c.  into  tube  No.  2. 

After  mixing  thoroughly  with  the  9  c.c.  of  water  in 
tube  2,  place  i  c.c.  of  the  mixture  in  plate  No.  2  and  i  c.c. 
into  tube  3.  Proceed  in  this  manner  through  the  series 
of  dishes.  Add  to  each  Petri  dish  10  c.c.  of  the  liquefied 
Hesse  agar,  cooled  to  40°  C.,  and  mix  thoroughly.  Cool 
in  the  ice  chest  to  set  the  medium,  and  incubate  24  hours 
at  37°  C. 

B.  typhosus  is  characteristic  on  Hesse  agar  only  when 
the  dilution  is  sufficiently  high  to  produce  but  a  few  bac- 
teria on  the  plate.  It  is  distinguished  from  B.  coli  by  the 
formation  of  colonies  of  much  larger  size,  and  consisting 
of  a  broad,  translucent,  or  scarcely  turbid  zone  between 
the  white  opaque  center,  or  nucleus,  and  the  circular, 
narrow,  white  seam,  or  edge. 

Potato  Slants 

Clean  Irish  potatoes  thoroughly  with  a  stiff  brush. 
Pare  off  the  outer  portion  and  cut  out  cylinders  with  a 
cork  borer,  |  inch  in  diameter,  then  divide  the  cylinders 
diagonally. 

Wash  in  running  water  several  hours. 

Place  a  small  piece  of  glass  tubing  in  the  bottom  of 
the  test  tube,  then  drop  in  the  potato,  base  downward. 
Sterilize  at  15  pounds  pressure  for  20  minutes. 

Glycerin  Potato 

Soak  the  potato  slant  in  6%  glycerin  for  i  hour.  Pro- 
ceed as  previously  directed. 


178  Clinical  Laboratory  Technic 

Special  Media  for  Rapid  Culture  of  Tubercle  Bacilli 
(Journal  of  Experimental  Medicine,  Vol.  XXI, 
No.  i) 

Two  parts  of  egg  (white  and  yolk). 

One  part  of  meat  juice. 

Gentian  violet  sufficient  to  the  proportion  of  i  to  10,000. 

MEAT  JUICE  :  500  grams  of  beef  or  veal  are  infused  in 
500  c.c.  of  a  15%  solution  of  glycerin  in  water.  Twenty- 
four  hours  later  the  meat  is  squeezed  through  a  sterile 
meat  press  and  collected  in  a  sterile  beaker. 

EGGS  :  Sterilize  the  shells  of  the  eggs  by  immersion  for 
10  minutes  in  70%  alcohol,  or  by  pouring  hot  water  upon 
them.  Break  the  eggs  into  a  sterile  beaker,  mix  well,  and 
filter  through  sterile  gauze.  Add  one  part  by  volume  of 
meat  juice. 

GENTIAN  VIOLET.  Add  sufficient  i%  alcoholic  gentian 
violet  to  make  a  dilution  of  TQIJO^- 

Tube  3  c.c.  in  sterile  test  tubes  and  inspissate  for  3 
successive  days.  On  the  first  day  at  85°  C,  until  all 
medium  is  solidified,  changing  the  places  of  the  tubes  if 
necessary;  on  the  second  and  third  days  for  not  more 
than  i  hour  at  75°  C. 

Differential  Medium  for  Acid-producing  Bacteria 
(Journal  A.  M.  A.,  Vol.  LXIV,  No.  6) 

Agar  medium  100  c.c. 

Milk  sugar  1.5  grams 

Congo  red  (aqueous  solution,  i%)        30  c.c. 


Culture  Media  179 

Typhoid  colonies  grown  on  this  media  are  red  and 
generally  transparent. 

Colon  colonies  develop  black,  with  a  light  halo. 

REFERENCES  :  Mallory  and  Wright's  Pathological  Tech- 
nique. Hiss  and  Zinsser's  Bacteriology.  Stitt's  Practical 
Bacteriology  and  Parasitology. 


CHAPTER  X 


BODY  FLUIDS 

Examination  of  body  fluids  is  useful  for  determining 
the  diagnosis  of  meningitis  and  the  differentiation  between 
inflammatory  fluid  and  hypostatic  fluid  in  cases  of  pleural 
or  peritoneal  effusion ;  i.  e.,  between  pleurisy  and  hydro- 
thorax,  peritonitis  and  ascites. 

Albumin  is  determined  by  the  methods  used  in  the 
examination  of  urine. 

Transudates  and  Exudates 

A  transudate  is  similar  to  lymph  and  collects  in  a  body 
cavity  on  account  of  various  circulatory  conditions. 

An  exudate  is  a  heavier,  more  cloudy  liquid,  which  is 
poured  out  upon  a  serous  or  other  surface  as  the  result 
of  inflammation. 

TRANSUDATE 

1.  Appearance:  Clear. 

2.  Clotting:   Almost  never 

spontaneously. 

3.  Sp.gr. :  Usually  less  than 


EXUDATE 

Clear  to  thick  and  creamy. 
Usually. 


i  ,020. 

4.  Albumin:  i%  or  less. 

5.  Nucleo-protoplasm : 

Little. 

6.  Sediment:  Slight,  endo- 

thelial  cells,  few  leuco- 
cytes. 


Usually  more  than  1,020. 

3%  or  over. 
Much  more. 


Considerable ; 
cytes. 


many  leuco- 


180 


Body  Fluids  181 

Cerebrospinal  Fluid 

1.  Amount:  5  to  10  c.c.  normally. 

2.  Clear. 

3.  Slightly  alkaline. 

4.  Specific  gravity:  1,005  to  1,010. 

5.  Protein,  nucleo-albumin,  etc. :  Considerable  amount. 

6.  Clotting :  It  may  on  standing. 

7.  Sediment:  Slight;  epithelial  or  endothelial  cells. 

Pericardial  Fluid 

1.  Amount:  Small. 

2.  Clear :  Normally. 

3.  Alkaline. 

4.  Protein :  High  per  cent,  and  contains  more  fibrin 
than  any  other  physiological  fluid. 

Synovial  Fluid 

1.  Color:  Yellow. 

2.  Appearance :    Thick,    viscid,    and    sticky ;    contains 
synovin,  a  mucin-like  body. 

3.  Protein :  Much. 

Pleural  Fluid 

NON-INFLAMMATORY  TRANSUDATE  :  Light  yellow,  clear, 
few  formed  elements.  Specific  gravity,  1,015  or  below. 

INFLAMMATORY  EXUDATE  :  Yellow,  clear  or  turbid  from 
fibrin,  and  formed  elements  present.  Specific  gravity, 
1,020. 

Purulent  Pleurisy 

Citron  color,  turbid.    Specific  gravity,  1,020. 
Albumin :  Large  amount. 


1 82  Clinical  Laboratory  Technic 

Cholesterin,  uric  acid,  bile  pigment,  and  sugar  often 
present. 

Cytodiagnosis 

The  examination  of  cellular  elements  of  fluid  with 
reference  to  the  variety  of  cell  which  predominates. 

Smears  are  made  from  the  sediment  and  stained  with 
Wright's  stain. 

Predominance  of  polymorphonuclear  leucocytes  means 
an  acute  infectious  process. 

Predominance  of  lymphocytes  means  tuberculosis. 

Few  cellular  elements  with  a  large  proportion  of 
endothelial  cells  means  a  transudate  or  mechanical 
effusion. 

Nonne's  Globulin  Test 

1.  i  c.c.  of  spinal  fluid. 

2.  i  c.c.  of  saturated  (NH4)2SO4. 

Pour  the  (NH4)2SO4  under  the  spinal  fluid  and  ex- 
amine for  white  ring  at  junction  of  fluids.  Faintest  possi- 
ble trace  equals  plus. 

Trace  with  black  background  equals  2  pluses. 

Trace  without  black  background  equals  3  pluses. 

Flocculent  precipitate  equals  4  pluses. 

If  blood  is  present,  a  pink  layer  usually  appears  in 
upper  stratum  of  globulin  test. 

Peritoneal  Fluid 

Normal  fluid  is   a  clear,  pale   straw  color.     Specific 
gravity,  1,005  to  T>OI5- 
Slightly  albuminous. 
Microscopically  it  shows  very  few  formed  elements. 


Body  Fluids  183 

Ascitic  Fluid 

Clear,  yellow,  albuminous;  coagulates  on  standing. 

In  ascites  adiposus  the  fluid  has  a  milky  appearance, 
due  to  the  presence  of  cells  that  have  undergone  a  fatty 
degeneration. 

In  ascites  chylosus  the  fluid  contains  chyle,  and  in 
pseudochylous  ascites  the  fluid  resembles  chyle  but  does 
not  contain  fatty  matter. 

Specific  gravity  is  about  1,015. 

Albumin,  2%. 

Microscopically  it  shows  few  leucocytes,  usually  fatty 
and  rarely  desquamated  epithelial  cells. 

Hydrocele  Spermatocele 

1.  Appearance:  Dark,  but      Colorless,  watery,  or  slightly 

clear.  milky. 

2.  Sp.  gr. :  1,014  to  1,026.         1,006  to  1,010. 

3.  Sometimes  coagulates         Contains  cell  detritus,    fat 

spontaneously.  globules,   and   spermato- 

zoa. 

The  fluid  should  be  collected  in  sterile  receptacles  under 
aseptic  precautions. 

Stain  smears  with  Loffler's  methylene  blue,  carbol  f  uch- 
sin  (Ziehl-Neelsen)  and  Gram's  stain. 

Cancerous  Fluids 

These  fluids  have  a  large  amount  of  albumin  and  a 
high  specific  gravity.  They  show  a  relatively  large  num- 
ber of  endothelial  cells  mixed  with  a  larger  percentage  of 
lymphocytes  than  are  found  in  the  mechanical  effusions. 


184  Clinical  Laboratory  Technic 

Cerebrospinal  Fluid  Examinations 

Make  culture  of  the  spinal  fluid  on  blood-serum  or 
blood-agar. 

The  cell  count  should  be  done  as  soon  as  possible. 
Normally  there  are  3  to  9  cells  per  cubic  millimeter.  In 
general  paresis  and  tabes  there  are  40  to  100  cells  per 
cubic  millimeter. 

An  excess  of  polymorphonuclear  and  eosinophilic  leu- 
cocytes1 is  indicative  of  meningococcic  or  pneumococcic 
infection,  and  a  lymphocytosis  indicates  a  tuberculous  or 
poliomyelitis  process. 

Counting  the  Cells 

Draw  up  Griibler's  polychrome  methylene  blue  in  a 
white  blood  cell  counter  to  the  mark  .5,  then  spinal  fluid 
up  to  ii.  Stain  3  minutes. 

Staining 

Centrifugalize,  and  drop  the  sediment  with  a  fine 
capillary  tube  at  intervals  on  the  slide ;  dry  and  fix  in  the 
flame.  Stain  with  Wright's  or  Gram's  stain. 

Butyric  Acid  Test 

Draw  up  .2  c.c.  of  fluid  in  a  sterile  graduated  pipette; 
add  .5  c.c.  of  a  10%  solution  of  butyric  acid  in  normal 
salt  solution  and  boil  I  minute,  then  add  .1  c.c.  of 
N/i  NaOH  and  boil  i  minute. 

A  precipitate  indicates  a  lesion. 

The  Lange  Colloidal  Gold  Test 

There  is  a  change  in  the  color  of  a  solution  of  colloidal 
gold  when  it  is  mixed  in  certain  proportions  with  the 

1  Also  streptococcic  and  influenzal  infection.  There  are  few 
cells  in  a  case  of  menineism.  Trypanosomiasis  gives  a  cellular 
increase  similar  to  syphilis. 


Body  Fluids  185 

cerebrospinal  fluid  from  cases  of  syphilis  and  certain 
other  pathological  conditions  of  the  central  nervous 
system. 

1.  Place  ii  tubes  in  a  rack. 

2.  To  the  first  tube  add  1.8  c.c.  of  a  0.4%  salt  solution 
from  a  burette. 

3.  To  each  of  the  following  tubes  add  i  c.c.  of  the 
0.4%  salt  solution. 

4.  Add  to  the  first  test  tube  0.2  c.c.  of  the  spinal  fluid, 
making  a  dilution  of  ^  using  a  sterile  pipette,  i  c.c. 
graduated  T^  to  the  tip. 

5.  Pipette  I  c.c.  from  this  tube  into  the  second  tube, 
mix  by  blowing,  and  so   on  to  each  of  the  ten  tubes. 
Tube    II    contains   no    spinal    fluid.      This    gives    dilu- 
tions of  the  spinal  fluid  of  &,  ^V,  $V»  sV>  Tao»   and  so  on 


6.  Add  5  c.c.  of  gold  reagent  to  each  tube  from  the 
burette,  without  shaking. 

The  results  are  read  in  12  to  24  hours. 

When  there  is  a  positive  reaction  the  gold  is  flaked  out, 
and  this  causes  a  color  change  which  varies  with  the  in- 
tensity of  the  reaction.  Color  in  the  Control  Tube  n 
should  be  salmon  red  and  the  fluid  should  be  trans- 
parent. The  changes  from  the  original  red  to  a  bluish 
tint  are  recorded  as  i.  When  the  change  is  to  a  lilac  it 
is  recorded  as  2.  A  distinct  blue  is  marked  as  3  and  a 
pale  blue  as  4.  Complete  discoloration  is  recorded  as 
5.  Minute  quantities  of  blood  do  not  affect  the  reaction, 
but  large  quantities  produce  unreliable  results. 

Normal  cerebrospinal  fluid,  free  from  serum  or  blood, 
gives  no  change  in  color. 


i86  Clinical  Laboratory  Technic 

Pathological  fluids  give  an  almost  immediate  change 
in  color. 

Non-specific  meningitis  and  brain  tumor  cases  show 
most  marked  changes  in  the  1-640  to  1-1,280  dilutions. 

Paretic  curve  (i-io  to  1-160). 

Cerebrospinal  lues  curve  (1-40  to  1-320). 

Meningitis  (1-320  to  1-2,560). 

Bacterial  contamination  of  the  fluid  may  change  a 
positive  reaction  into  a  negative  one. 

Preparation  of  Colloidal  Gold  Solution 

New  glassware  is  preferable  and  should  not  be  used 
for  anything  else. 

Clean  glassware  with  hot  soapy  water,  rinse  with  aqua 
regia,  then  thoroughly  rinse  with  tap  water  several  times. 
Rinse  again  with  doubly  distilled  water ;  sterilize. 

The  distilled  water  used  must  be  doubly  distilled  from 
Jena  glass  in  a  still  free  from  rubber  connections,  and  it 
must  be  kept  in  Jena  glass  flasks. 

I  liter  of  doubly  distilled  water ;  heat  on  slow  flame  to 
60°  C.  Keep  thermometer  in  the  flask.  When  the  tem- 
perature is  reached,  add  10  c.c.  of  a  i%  aqueous  solution 
of  gold  chloride  (Merck's  15  grains  ampules  to  100  c.c.  of 
water;  keep  in  a  brown  bottle). 

10  c.c.  of  a  2%  aqueous  solution  of  K2CO3. 

Add  solutions  simultaneously.  Heat  rapidly  to  just 
under  boiling.  Remove  from  flame  and  add  immediately 
10  c.c.  of  a  i%  aqueous  solution  of  40%  formalin.  Shake, 
and  continue  to  shake  until  proper  color  develops.  If  it 
does  not  develop,  throw  away  and  start  over  again.  Solu- 
tion should  be  red,  perfectly  clear  by  transmitted  light, 
and  opalescent  by  reflected  light. 


CHAPTER  XI 


MILK 


Composition 

HUMAN  MILK 


Cow's  MILK 


Fat 

3  to  4% 

Fat 

Lactose 

5  to  8% 

Lactose 

Protein 

I    tO    2% 

Protein 

Ash 

O.2  tO  0.4% 

Ash 

Water 

87  to  88% 

Water 

3.5% 
4.5% 

3-9% 
0.7% 

874% 


Reaction 

HUMAN  MILK 
Amphoteric. 

Specific  Gravity 

HUMAN  MILK 
1,028  to  1,032. 


Cow's  MILK 
Slightly  alkaline. 


Cow's  MILK 


1,031. 


Definition  of  Terms 

SEPARATED  MILK  :  Milk  from  which  fat  has  been  par- 
tially or  wholly  removed,  either  by  centrifuge  or  gravity. 

FAT  FREE  :  Separated  milk  which  contains  no  fat,  or  at 
least  a  fractional  per  cent  such  as  may  be  obtained  in  the 
lowest  quarter  of  a  quart  of  milk  which  has  been  setting 
6  or  more  hours. 

CREAM  :  That  which  remains  after  separated  milk  has 
been  removed  from  whole  milk.  It  contains  most  of  the 

187 


1 88  Clinical  Laboratory  Te clinic 

fat  of  whole  milk  and  certain  percentages  of  all  the  other 
elements  of  whole  milk. 

WHOLE  MILK  :  This  is  considered  4%  cream. 

WHEY  :  Contains  all  the  sugar  and  water  of  milk,  while 
most  of  the  fat  and  all  caseinogen  have  been  removed. 

CASEINOGEN:  Mother  substance,  from  which  casein  is 
obtained  by  precipitation  or  coagulation. 

Fermentation 

Place  milk  in  a  warm  room  and  it  becomes  acid.  This 
is  due  to  the  action  of  bacterium  lactis,  which  changes 
the  milk-sugar  (lactose)  into  lactic  acid  and  precipitates 
the  casein. 

Bacillus  aerogenes  capsulatus  and  bacillus  odematis 
maligni  produce  butyric  acid  fermentation. 

Yeasts  produce  alcoholic  fermentation. 

The  chromogenic  bacteria,  B.  cyanogenus,  B.  prodigi- 
osus,  and  others,  produce  the  blue,  green,  and  yellow 
changes  which  sometimes  occur  in  milk. 

Strepto-lacticus  is  found  in  sour  milk. 

Milk  containing  over  1,000,000  bacteria  to  the  c.c.  is 
considered  harmful  to  the  infant. 

Methods  of  Preservation. — Refrigeration 

Milk  kept  at  or  below  10°  C.  (50°  F.)  will  remain 
sweet  and  uncurdled  for  several  days ;  but  cold  will  not 
preserve  milk  indefinitely,  nor  will  it  kill  bacteria. 

Sterilization 

If  fresh  milk  is  boiled  15  minutes,  a  scum  is  formed, 
due  to  the  coagulation  of  lactalbumin  and  globulin.  This 
kills  the  bacteria  which  cause  lactic  fermentation,  and 


Milk  189 

the  milk  will  remain  sweet  for  several  days  if  the  recep- 
tacle is  stoppered  with  cotton.  Boiled  milk  is  less  digesti- 
ble and  nourishing  as  the  fats,  sugars,  casein,  and  albumin 
are  altered.  Milk  is  rendered  germless  when  heated  at  a 
temperature  of  68°  to  75°  C.  (154°  to  167°  F.),  and  the 
taste  and  digestibility  are  not  materially  altered.  A  tem- 
perature of  68°  C.  is  sufficient  for  the  destruction  of 
tubercle  bacilli ;  60°  C.  for  typhoid ;  58°  C.  for  diphtheria ; 
and  most  saprophytes  will  be  killed  at  a  temperature  of 
75°  C. 

Pasteurization 

This  method  is  preferred  to  complete  sterilization,  as 
it  does  not  affect  materially  the  nutritive  value  of  milk. 
Pathogenic  bacteria  and  non-pathogenic  micro-organisms 
that  cause  the  souring  of  milk  are  destroyed.  Spores  are 
not  destroyed  by  this  process.  Place  the  fresh  milk  in 
sterile  glass  bottles,  stopper  with  cfltton  and  stand  in  a 
vessel  of  water  (a  double  boiler),  and  heat  the  water  to 
70°  C.  for  |  hour.  The  milk  will  remain  sweet  24  hours. 

Test  for  Pasteurized  or  Sterilized  Milk 

1.  Place  10  c.c.  of  milk  in  a  test  tube  and  heat  to  70°  C. 

2.  Place  10  c.c.  of  milk  in  a  second  test  tube  and  heat 
to  80°  C. 

3.  Cool,  and  add  to  each  i  c.c.  of  paraphenylene  di- 
amine  solution ;  add  a  few  drops  of  hydrogen  peroxide. 
There   is   an   instantaneous  change  to   deep  blue   color, 
caused  by  the  unchanged  enzymes  in  the  first  tube ;  while 
the  overheated  milk  in  the  second  tube  does  not  change 
to  the  blue  color  for  some  time. 

Other  preservatives  used  in  connection  with  milk  are 


190  Clinical  Laboratory  Technic 

boric   acid,   borates,    formaldehyde,   hydrogen   peroxide, 
salicylic  acid,  and  salicylates. 

Test  for  Boric  Acid  and  Borates 

Place  2  c.c.  of  the  milk  in  a  porcelain  dish  and  expel 
the  water  by  heating  over  a  water  bath.  Then  heat  the 
solids  over  a  low  flame  until  a  white  or  light  gray  ash 
is  obtained.  Add  2  drops  of  dilute  HC1  acid  in  i  c.c.  of 
water.  Soak  a  piece  of  turmeric  paper  in  a  dish,  then 
remove  and  dry  in  the  air.  Boric  acid  is  indicated  by  a 
deep  red  color,  which  changes  to  green  or  blue  upon  add- 
ing dilute  alkali. 

Formaldehyde. — Leach's  Test 

Mix  10  c.c.  of  the  milk  and  10  c.c.  of  concentrated  HC1 
containing  0.002  gram  ferric  chloride  in  a  porcelain  dish, 
and  gradually  raise  the  temperature  of  the  mixture  on  a 
water  bath  nearly  t*o  boiling,  with  occasional  stirring. 
A  violet  color  is  produced  if  formaldehyde  is  present. 

Hydrogen  Peroxide 

To  10  to  15  c.c.  of  milk  add  3  drops  of  a  2%  aqueous 
solution  of  paraphenylenediamine  hydrochloride.  Shake. 
A  blue  color  appears  immediately  in  the  presence  of 
hydrogen  peroxide. 

Salicylic. — Remont's  Method 

Acidify  20  c.c.  of  milk  with  sulphuric  acid.  Shake 
vigorously  to  break  up  the  curd.  Add  25  c.c.  of  ether, 
mix,  and  decant  5  c.c.  of  the  ethereal  extract ;  evaporate 
to  dryness. 

Boil  the  residue  with  10  c.c.  40%  alcohol.     Cool,  and 


Milk  191 

make  up  the  volume  to  10  c.c.  Filter,  and  to  5  c.c.  of 
filtrate,  which  represents  2  c.c.  of  milk,  add  2  c.c.  of  a 
2%  solution  of  ferric  chloride.  A  purple  or  violet  color 
indicates  the  presence  of  salicylates. 

Fat' 

To  5  c.c.  of  milk  in  a  small  Babcock  tube  add  an  equal 
volume  of  sulphuric  acid  (sp.  gr.,  1.83)  and  enough  of 
a  mixture  of  concentrated  HC1  and  amyl  alcohol  to  fill 
the  neck  of  the  tube.  Centrifugalize  5  minutes.  The 
per  cent  of  fat  is  read,  off  directly  on  the  tube,  and  is 
accurate  to  within  0.5%.  If  the  top  of  the  fat  column 
is  not  at  zero,  add  water  and  centrifugalize  I  minute. 

Counting  Bacteria 

Mix  the  contents  of  the  can,  as  the  cream  contains 
more  bacteria  than  the  milk.  Place  the  specimen  in  a 
sterile  test  tube.  With  a  sterile  pipette  dilute  the  milk  in 
sterile  water.  Inoculate  cooled  tubes  of  melted  agar  with 
varying  amounts  of  these  dilutions  and  pour  on  sterile 
Petri  plates.  Incubate  12  to  24  hours,  then  count  colonies. 

EXAMPLE.  Add  i  c.c  of  milk  to  199  c.c.  of  sterile 
water.  Mix  thoroughly.  Then  take  I  c.c.  of  this  1-200 
dilution  and  add  it  to  99  c.c.  of  sterile  water.  Mix  thor- 
oughly. This  makes  a  1-20,000  dilution.  Add  0.5  c.c. 
of  this  to  a  plate  of  gelatin  or  agar.  Incubate.  If  50 
colonies  were  counted  after  incubation  the  milk  contained 
in  each  cubic  centimeter  50  X  2  X  20,000,  or  2,000,000 
— the  number  of  bacteria  in  each  c.c.  of  milk.  , 

If  typhoid  is  suspected,  use  same  media  and  method 
(Hesse)  as  in  detecting  typhoid  in  feces. 

REFERENCES:  Medical  Chemistry  and  Toxicology, 
Holland.  Physiological  Chemistry,  Hawk. 


CHAPTER  XII 
PREPARATION  OF  TISSUE 

The  specimen  should  not  be  larger  than  ^  inch  square, 
and  this  should  be  placed  in  fixing  solution  as  soon  as 
possible.  Zenker's  fluid  is  considered  the  best  general 
fixative  for  histological  study,  as  the  bacteria,  nuclear 
figures,  and  fibrils  of  all  kinds,  are  better  preserved. 
Tissues  fixed  in  Zenker's  fluid  and  corrosive  sublimate 
must  be  transferred  after  thorough  washing  in  water  to 
70%  alcohol,  then  So%  alcohol. 

Tissues  hardened  in  alcohol  and  formaldehyde  may 
remain  indefinitely  in  those  fluids;  and  tissue  fixed  by 
alcohol,  or  any  other  reagent  except  formaldehyde,  must 
be  washed  in  running  water  several  hours  before  freezing. 

Tissue  fixed  in  formaldehyde,  wash  20  to  30  minutes. 

Freezing   Method  for  Rush  Diagnosis 

The  microtome  is  fastened  firmly  in  position  and  the 
wire  attached.  Connect  the  other  end  of  the  wire  to 
the  tube  of  carbon  dioxide. 

Place  a  piece  of  the  tissue  on  the  freezing  box  of  the 
microtome,  with  a  few  drops  of  water  beneath  it.  Turn 
the  gas  on  slowly  at  first,  then  rapidly. 

Hold  the  handle  of  the  knife  so  that  the  thumb  presses 
against  the  end  of  the  wooden  part,  and  apply  the  edge, 
bevel  side  downward,  at  an  angle  of  45°. 

Turn  the  wheel  of  the  microtome  screw  with  the  other 

Note.  For  myelogenous  leukemia  tissue  use  Heller's  solution 
(95  c.c.  of  Zenker's  solution  without  the  acetic  acid,  and  5  c.c. 
of  formalin). 

192 


Preparation  of  Tissue  193 

hand,  and  cut  several  sections  without  changing  the 
position  of  the  hand  or  the  angle  of  the  knife.  Place 
the  cut  sections  in  a  small  pan  of  water.  Clean  a  slide  by 
rubbing  it  with  alcohol,  then  spread  a  thin  layer  of  albu- 
min mixture  on  the  surface  with  a  fine  brush,  and  rub 
this  in  thoroughly  with  the  finger,  having  previously 
cleaned  the  finger  by  wiping  it  with  alcohol. 

Dip  the  slide  into  the  dish  under  the  section  so  that 
it  can  be  floated  on  and  spread  out  evenly  on  its  surface. 

Drain  and  blot  with  a  smooth  blotting  paper.  Cover 
with  95%  alcohol,  drain,  and  cover  with  absolute  alcohol. 
Drain  and  cover  with  a  thin  solution  of  collodion.  Drain, 
and  if  the  tissue  is  to  be  examined  for  rush  diagnosis, 
cover  with  Loffler's  methylene  blue  (dilute  J)  for  15 
seconds. 

Delafield's  Alum-Hematoxylin  Method 

After  fixing  the  tissue  on  the  slide  in  the  manner 
described  above,  cover  with  alum-hematoxylin  stain 
3  to  5  minutes.  Wash  in  several  changes  of  water. 

Cover  with  contrast  stain  of  eosin  (aqueous  solution 
of  7*0  to  %%)  2  to  5  minutes. 

Remove  the  excess  of  stain  by  three  changes  of  95% 
alcohol. 

Clear  in  oleum  origani  cretici.    Mount  in  xylol  balsam. 

If  the  oleum  origani  is  not  used,  remove  the  excess  of 
stain  with  absolute  alcohol,  then  clear  with  xylol,  and 
mount  in  xylol  balsam. 

Satisfactory  sections  are  obtained  from  fresh  tissue 
frozen  in  this  manner,  but  the  formation  of  ice  crystals 
frequently  causes  tearing  of  delicate  tissue,  like  the  brain, 
spinal  cord,  and  retina;  so  that  it  is  better  to  infiltrate 


194  Clinical  Laboratory  Technic 

such  tissues  with  a  mass  that  does  not  crystallize  in  the 
freezing  mixture,  but  becomes  hard  and  tough. 

The  delicate  tissue  is  soaked  in  a  sirup  made  by  dissolv- 
ing i  pound  of  loaf  sugar  in  i  pint  of  water  and  boiling. 

Remove  the  sirup  from  the  outside  of  the  tissue  with 
a  cloth  and  put  it  into  ordinary  gum  mucilage  for  i  hour, 
then  place  on  the  microtome  and  freeze  in  the  usual 
manner. 

Mayer's  Albumin  Solution 

White  of  i  egg  25  c.c. 

Glycerin  25  c.c. 

Salicylate  of  soda  0.5  gram 

Water  4  c.c. 

Dissolve  the  salicylate  in  the  water,  then  add  the  white 
of  egg  and  beat  thoroughly.  Add  the  glycerin  and  filter. 

Collodion  Mixture 

Flexible  collodion  I  c.c. 

Absolute  alcohol  15  c.c. 

Ether  15  c.c. 

It  is  necessary  to  imbed  the  sections  in  celloidin  or 
paraffin  for  finer  histological  work,  and  for  the  highest 
class  of  work  in  tissue  cutting  it  is  necessary  to  have  a 
microtome  in  which  the  knife  is  a  fixture.  This  process 
is  a  much  longer  one  than  the  freezing  method,  and  takes 
from  24  to  36  hours. 

Fresh  tissue  may  also  be  examined  in  teased  prepara- 
tions made  by  cutting  a  very  small  section  and  dividing 
it  by  means  of  two  sharp  needles.  Place  on  a  slide  in  a 
drop  of  normal  salt  solution.  Cells  may  be  easily  obtained, 


Preparation  of  Tissue  195 

if  the  tissue  is  soft,  by  scraping  the  cut  surface  with  the 
edge  of  a  knife. 

Decalcification 

Place  a  small  piece  of  bone  in  formalin  (10%)  for 
24  hours,  then  transfer  to  concentrated  sulphuric  acid 
for  24  hours.  Wash  thoroughly  in  alkaline  water  and 
then  in  tap  water.  Proceed  as  with  other  frozen  sections. 

Nicolle's  Method 

1.  Loffler's  methylene  blue,  10  minutes. 

2.  Acetic  acid  (1-500),  10  seconds. 

3.  Tannin  (i%),  5  seconds. 

4.  Wash  in  water,  95%  alcohol,  absolute  alcohol,  and 
xylol.    Mount  in  balsam. 

Van  Gieson's  Stain 

1.  Stain  deeply  in  alum-hematoxylin. 

2.  Wash  in  water. 

3.  Stain  in  Van  Gieson's  solution  3  to  5  minutes. 

4.  Wash  in  water. 

5.  Dehydrate  in  95%  alcohol. 

6.  Cover  with  oleum  origani  cretici. 

Zenker's  Fluid 

Bichromate  of  potassium  12.5  grams 

Corrosive  sublimate  25  grams 

Water  500  c.c. 

Dissolve  the  corrosive  sublimate  and  bichromate  in 
water  with  the  aid  of  heat.  To  every  50  c.c.  of  Zenker's 
solution,  which  is  sufficient  for  one  specimen,  add  2.5  c.c. 
of  glacial  acetic  acid  before  dropping  in  the  tissue.  Fixa- 
tion is  accomplished  in  24  hours. 


196  Clinical  Laboratory  Technlc 

Mounting 

Xylol  balsam  is  the  best  reagent  used  for  permanent 
mounts.  It  has  a  high  index  of  refraction,  and  tissues 
mounted  in  it  become  very  transparent. 

To  clean  the  microtome  wipe  with  alcohol,  then  ether, 
and  lastly  wipe  with  an  oily  cloth. 

REFERENCES  :  Mallory  and  Wright's  Pathological  Tech- 
nic.  Stohr's  Textbook  of  Histology.  Lee's  The  Micro- 
tomist's  Vade-Mecum. 


APPENDIX 

Reagents  for  Blood 

Gower's  Diluting  Solution  for  Counting  Red  Blood 
Cells 

Sodium  sulphate  7.5  grams 

Acetic  acid  20.0  c.c. 

Aqua  128  c.c. 

Haymen's  Solution  for  Counting  Red  Blood  Cells 
Mercuric  bichloride  0.25  gram 

Sodium  sulphate  2.5  grams 

Sodium  chloride  0.5  gram 

Distilled  water  100  c.c. 

Diluting  Solution  for  Counting  White  Blood  Cells 
Acetic  acid  I  c.c. 

Distilled  water  300  c.c. 

Toisson's  Solution  for  Simultaneously  Counting  Red 
and  White  Cells 

Methyl  violet  0.05  gram 

Neutral  glycerin  30  c.c. 

Distilled  water  80  c.c. 

Mix  and  add : 

Sodium  chloride  i.oo  gram 

Sodium  sulphate  8.00  gram 

Distilled  water  80  c.c. 

Filter.    12  minutes  required  to  stain  white  blood  cells. 


1 98  Clinical  Laboratory  Technic 

Wright's  Stain 

Sodium  bicarbonate  (C.  P.)  0.5  gram 

Distilled  water  100  c.c. 

Methylene  blue  (B.  X.)  I  gram 

Sterilize  I  hour  at  15  pounds  pressure.  When  cold 
add  500  c.c.  of  ToW  solution  Eosin  Gruber  w.  g.,  or 
enough  to  make  the  mixture  purple  with  a  metallic  scum 
on  the  surface.  The  precipitate  is  then  collected  on  a  filter 
and  allowed  to  dry.  When  thoroughly  dry,  dissolve  this 
precipitate,  0.5  gram  in  methyl  alcohol,  Merck  reagent, 
100  c.c.,  which  makes  the  stain. 

Reagents  for  Urine 
Nitric  acid,  (HNO3). 
Acetic  acid,  (HC2H3O2). 
Sulphuric  acid,  C.  P.  (H2SO4). 
Hydrochloric  acid,  (HC1)  C.  P. 
Ammonic  hydrate,  (NH4OH). 
Sodic  hydrate,  (NaOH),  U.  S.  P. 

Esbach's  Reagent 

Picric  acid  10  grams 

Citric  acid  20  grams 

Water  1,000  c.c. 

Fehling's  Reagent 

Solution  "A": 

Copper  sulphate  34-^5  grams 

Distilled  water  500  c.c. 

Solution  "B": 

Sodium  potassium  tartrate  173  grams 

Sodium  hydroxide  125  grams 

Water  500  c.c. 


Appendix  199 

Magnesia  Mixture 

Ammonium  chloride  I  part 

Magnesium  sulphate  I  part 

Ammonia  water  I  part 

Water  8  parts 
Dissolve  the  salts  in  the  water,  then  add  the  ammonia 

water. 

Nylander's  Reagent 

Bismuth  subnitrate  2  grams 

Rochelle  salt  4  grams 

Sodium  hydroxide  (8%)  IOO  c.c. 

Phosphotungstic  Solution 

Phosphotungstic  1.5  grams 

Hydrochloric  acid  5  c.c. 

Alcohol  100  c.c. 

Standard  Potassium  Sulphocyanide  (KCNS) 
Solution 

Potassium  sulphocyanide  8.29  grams 

Distilled  water  to  1,000  c.c. 

2  c.c.  corresponds  to  I  c.c.  of  standard  silver  nitrate 
solution. 

Sodium  Acetate  Solution 

Sodium  acetate  100  grams 

Distilled  water  800  c.c. 

Dissolve  this  and  add  100  c.c.  of  30%  acetic  acid.    Make 

up  to  i  liter. 

Uranium  Acetate  Solution 

Uranium  acetate  34  grams 

Distilled  water  1,000  c.c. 

i  c.c.  equals  0.005  gram  P2O5,  phosphoric  anhydride. 


2OO  Clinical  Laboratory  Technic 

To  STANDARDIZE  :  To  50  c.c.  of  a  standard  solution  of 
disodium  hydrogen  phosphate,  of  such  strength  that 
50  c.c.  contains  o.i  gram  P2O5,  add  5  c.c.  of  sodium 
acetate  solution  and  titrate  with  the  uranium  solution  to 
the  correct  end  reaction. 

i  c.c.  of  uranium  solution  should  precipitate  0.005  gram 
of  P205. 

20  c.c.  of  uranium  solution  should  be  required  to  pre- 
cipitate 50  c.c.  of  the  standard  phosphate  solution. 

Standard  Ammonium  Thiocyanate  Solution 

(i  c.c.  equals  i  c.c.  standard  silver  nitrate) 
Ammonium  thiocyanate  12.9  grams 

Distilled  water  800  c.c. 

Place  20  c.c.  of  standard  silver  nitrate  solution  in  a  flask, 
5  c.c.  of  a  cold  saturated  solution  of  ferric  alum,  and  4  c.c. 
of  nitric  acid  (sp.  gr.,  1.2).  Add  water  to  100  c.c.  Mix. 
Fill  burette  with  the  ammonium  thiocyanate  solution,  run 
in  until  a  permanent  red-brown  color  is  obtained,  which 
is  the  end  point  and  indicates  that  the  last  trace  of  silver 
nitrate  has  been  precipitated.  Make  a  reading  and  calcu- 
late the  amount  of  water  necessary  to  use  in  diluting  the 
ammonium  thiocyanate  so  that  10  c.c.  may  equal  10  c.c.  of 
the  silver  nitrate.  Make  the  dilution  and  titrate  again. 

Standard  Silver  Solution 

Silver  nitrate  29.042  grams 

Distilled  water  to  make  1,000  c.c. 

i  c.c.  equals  o.oi  gram  of  sodium  chloride,  or  0.006  gram 
chlorine. 
INDICATOR  : 

Ferric  ammonium  sulphate  crystals      100  grams 
Dissolved  in  25%  nitric  acid  100  c.c. 


Appendix  201 

Standard  Solution  Uranium  Nitrate 

Uranium  nitrate  44.8  grams 

Distilled  water  to  make  900  c.c. 

Titrate  the  solution  with  a  standard  phosphate  solution ; 
the  amount  of  water  to  be  added  to  the  remainder  of  the 
uranium  solution,  so  that  I  c.c.  will  be  equivalent  to 
the  0.005  gram  of  P2O5,  can  be  calculated. 

Ammonium  Sulphocyanate  Solution 

Ammonium  sulphocyanate  13  grams 

Distilled  water  800  c.c. 

Titrate  this  solution  against  the  standard  nitrate  and 
estimate  how  much  water  should  be  added  to  the  re- 
mainder to  make  it  exactly  equivalent  to  the  standard 
silver  solution. 

Roberts'  Reagent 

Concentrated  HNO3  I  c.c. 

Saturated  solution  of  MgSO4  5  c.c. 

Reagents  for  Gastric  Analysis 

Congo  Red  Solution 

Congo  red  0.5  gram 

Alcohol  (95%)  10  c.c. 

Water  ,  90  c.c. 

Dissolve  the  dye  in  the  water  and  add  the  alcohol. 

Resorcin  Solution 

Resorcin  5  grams 

Cane  sugar  3  grams 

Alcohol  (95%)  ioo  c.c. 


2O2  Clinical  Laboratory  Technic 

Uffelmann's  Reagent 

Carbolic  acid  (4%)  10  c.c. 

Water  20  c.c. 

Ferric  chloride  solution  (U.  S.  P.)     i  drop 
(This  solution  should  be  prepared  fresh  for  use.) 

Gunzburg's  Reagent 

Phloroglucin  2  grams 

Vanillin  I  gram 

Alcohol  30  c.c. 

(Keep  in  colored  bottle.) 

Lugol's  Iodine  Solution 

Iodine  I  part 

Potassium  iodide  2  parts 

Water  50  parts 

Reagents  for  Stains 

Gram's  Stain. — No.  1 

Gentian  violet  2  grams 

Aniline  oil  9  c.c. 

Alcohol  (95%)  33  c.c. 

No.  2 

Gentian  violet  2  grams 

Distilled  water  100  c.c. 

Mix  i  c.c.  of  No.  i  with  9  c.c.  of  No.  .2 ;  filter.     This 
solution  will  not  keep  longer  than  three  weeks. 

Gram's  Iodine  Solution 

Iodine  i  gram 

Potassium  iodide  2  grams 

Distilled  water  300  c.c. 


Appendix  203 

Loffler's  Methylene  Blue 

Saturated  alcoholic  methylene  blue     30  c.c. 
Solution  KOH,  T^OTT  IQo  c.c. 

The  dilute  KOH  solution  may  be  made  by  adding  i  c.c. 
of  a  i%  solution  to  99  c.c.  of  water. 

Neisser's  Stain. — No.  1 

Methylene  blue  o.i  gram 

Alcohol  (95%)  2  c.c. 

Glacial  acetic  acid  5  c.c. 

Distilled  water  95  c.c. 
Dissolve  the  methylene  blue  in  the  alcohol,  then  add 

the  acetic  acid  and  water,  and  filter. 

No.  I 

Bismarck  brown  0.2  gram 

Water  (boiling)  100  c.c. 

Dissolve  the  dye  in  the  boiling  water  and  filter. 

Carbol  Fuchsin 

Basic  fuchsin  I  gram 

Alcohol  10  c.c. 

Carbolic  acid  (1-20)  100  c.c. 

Gabbet's  Stain 

Methylene  blue  2  grams 

Sulphuric  acid  25  c.c. 

Water  .  75  c.c. 


2O4  Clinical  Laboratory  Technic 

Giemsa's  Stain 

Azur  II  eosin  0.3  gram 

Azur  II  0.08  gram 

Glycerin,  C.  P.  25  c.c. 

Methyl  alcohol  25  c.c. 

Dissolve  the  dye  in  the  glycerin  at  60°  C.,  then  add 

the  methyl  alcohol  at  the  same  temperature. 

Aqueous  Alum-Hematoxylin  Stain 

Hematoxylin  crystals  i  gram 

Saturated   aqueous   solution   of 

ammonia  alum  100  c.c. 

Water  300  c.c. 

Thymol  I  crystal 

Dissolve  the  hematoxylin  crystals  in  water  with  heat. 
Expose  to  the  light  in  a  bottle  lightly  stoppered.  *  After 
10  days  keep  in  a  tightly  stoppered  bottle. 

Delafield's  Hematoxylin 

Hematoxylin  crystals  4  grams 

Alcohol  (95%)  25  c.c. 

Saturated   aqueous   solution   of 

ammonia  alum  400  c.c. 

The  hematoxylin  is  dissolved  in  the  alcohol  and  added 
to  the  alum  solution.  Expose  the  mixture  in  an  un- 
stoppered  bottle  for  4  days.  Filter  and  add : 

Glycerin  100  c.c. 

Alcohol  (95%)  loo  c.c. 

Keep  in  the  light  until  the  color  is  dark,  then  filter  and 
keep  in  a  tightly  stoppered  bottle.  Dilute  10  to  15  times 
when  staining. 


Appendix  205 

Polychrome  Methylene  Blue 

This  stain  is  an  old  alkaline  solution  of  methylene  blue 
and  carbonate  of  potassium  in  which,  as  a  result  of  oxida- 
tion, methyl  violet  and  methylene  red  form. 

The  process  of  oxidation  requires  months.  A  ripened 
solution  may  be  obtained  from  Grubler. 


INDEX 


Acetone,   34 
Achlorhydria,    71 
Achromia,   92,    105 
Achylia   gastrica,   71 
Acid,  acetic,  91,  98 

butyric,    1 84 

combined,  81 

fatty,   58 

free  hydrochloric,  71,  72,  81 

lactic,  72,  73 

nitric,    75 

organic,   72,  82 

oxalic,  75 

uric,   37 

Acidity  of  gastric  contents, 
quantitative  estimation 
of,  78-80 

urine,  cause  of,  23 
Aerobes,   144 

Aerogenes  capsulatus,   150 
Agar-agar  media,   169 
Agglutination,  115,   116 
Agglutinins,    128 
Albumin,   in  body  fluids,    180 

in  urine,  25,  26,  27,  28 
Albuminometer,  27 
Albuminuria,  25 
Alcohol,  6 

Alkaline  -  eaction,   in  urine,   23 
Alkaline  tide,  23 
Alkaloids,   76 
Alkapton,   23 
Alum-Hematoxylin    method,     193 

stain,  preparation  of,  204 
Amboceptor,    126,   128 

slips,    127 
Ameba  coli,   63 

histolytica,  63 

tetragena,   63 
Ammonia,  test  for,  49 
Ammonium,    sulphocyanate    solu- 
tion, 201 

thiocyanate    solution,   200 
Amorphous  phosphates,  in  urine, 

39  f 
urates,  in  urine,  40 


Amphoteric  reaction,  24 
Anaerobes,   145 
Anemia,  107 

pernicious,   108 
Animal  parasites,  in  feces,  63-65 

in  urinary  sediment,  45 
Anisocytosis,   92 
Antibodies,    128 

Anti-complementary  action,   128 
Antigen,    124,    128 
Appendix,    197 
Arsenic,  74 
Ascitic  fluid,   183 
Ash  of  milk,   187,  190 
Atomic  weights,  8 

Babcock   fat  method,  191 
Bacilli,  acne,    151 

aerogenes   capsulatus,   151 

anthracis,   151 

coli    communis,    116,    149 

diphtheria,   150,    154,    156 

diphtheroids,    154 

Gram    negative,    154 
positive,    154 

influenza,    144,    150 

leprosy,    156 

para-typhoid,    "type  A,"    116 
"type   ri,"   116 

pyocyaneus,    151 

smegma,    156 

tubercle,  46,   60,   86,  150,    156 

typhoid,  1 08,   no,   149 
Bacteria,  acid,   142 

aerobic,    144 

anaerobic,    145 

Pasteur's     method    of    culti- 
vating,   145 
Wright's   method,    145 

Brownian    movement,    143 

chemical    composition,    141 

cilia,    143 

color,    142 

counting,    146 

culture  examination,    144 

descriptive  chart,    161 


206 


Index 


207 


Bacteria   distribution   in  the  ani- 
mal body,   1 60 

endospores,    144 

ferments,    142 

flagella,    143 

form,    141 

gas,   142 

isolating,   147 

lesions   caused   by,   151 

size,    142 

spores,   144 

staining  methods,    152 
Bacterial  vaccines,   145 
Balance,   2,   3 
Basophile,    106 
Bence-Jones  bodies,  26 
Benedict's  tests,  29,  31 
Benzidin   reaction   for   blood,    in 
feces,  59 

in  gastric  contents,   74. 

in  urine,  34 
Bile  pigment,  in  faeces,  60 

in  urine,   36 
Bilharzia  hematobia,  46 
Bilirubin,  in  feces,  60 

in   urine,    36 
Bismarck  brown,   6 
Blood,  agglutination,  115,  116 

amount,    91 

bacteria   in,    95 

chemistry,   132 

coagulation,    time,    96 

color,    90 

color  index,  96 

counters,  97 

counting,    diluting   solution 

for,   197 

red  blood  cells,  98,    101 
white  blood  cells,  98,  100 

counting  chamber,   99 

determination     of     non-protein 
nitrogen,    132 

differential   count,    103 

diseases,  107-109 

dust,  95 

erythrocytes,   91,   92,    104 
counting   of,    101 

fragility,   no 

function,  92 

groups,   113,    114 

hemoglobin,   Sahli's  method,  96 
Talquist's,    97 


Blood  hemoglobinometer,  96 
hemolysis,  91 
laked,  91 
leucocytes,   94 

counting  of,    100 

in  urine,  43 

macroscopic   test,   114 
odor,  90 

opsonic   index,  95 
parasites,    121 
plasma,  95 
platelets,    114,    115 
pressure,   130 

auscultatory    method,    131 

diastolic,    130 

factors   influencing,    132 

mean,   131 

palpatory   method,    131 

systolic,    130 
reaction,  91 
reticulated,  103 
serum,   95,   170,  173 
smears,    103 

specific  gravity,  91  ^ 

staining  methods,  102,  103 
taste,    90 

vital  staining,   102 
urea,    134 

Wassermann's   reaction,    121 
Body  fluids,   180 
Bouillon,    169 
dextrose,    171 
glucose,    169 
sugar,    174 
sugar  free,   170,  174 
Burnham's     test     for     formalde- 
hyde,  46 
Butyric  acid,  test  for,  184 

Cabot's  rings,  93 

Calcium     carbonate    crystals,    38, 

39 
Calcium  oxalate  crystals,  38,  39, 

4i 

Calculi,  urinary,  41 
Cancerous   fluids,    183 
Carbohydrates,  in  feces,  59 

in  urine,   28 
Carbol  fuchsin,  203 
Carbolic  acid,  74 
Carbon  dioxide,   192 
Caseinogen,   188 


208 


Index 


Casts,  43 

blood,  44,  45 

epithelial,  44 

fatty,  45 

granular,   44 

hyaline,  43 

waxy,  45 
Caudate  cells,  42 
Centigrade  scale,  13,  14 
Cerebro-spinal  fluid,   181,   184 
Charting  uniform,   47 
Chloral,  75 
Chlorides  in  urine,  quantitative 

determination  of,  48 
Cholesterin  crystals,  38,  41 
Chlorosis,  108 

Cleaning    fluid    for    glass,    prep- 
aration of,  2 

Coagulation  method  of  determin- 
ing r  te,  96 

test,  26 

time,   96 

Collodion  mixture,  194 
Colloidal  gold  test,   184 
Common  elements,  9 
Complement,  129 

slips,   126 
Congo  red  paper  test,  71 

solution,  preparation  of,  201 
Counting  chamber,  for  blood,  99 

for  bacteria,  146 

for  cells,   184 
Creatinine,   37 
Crenated  cells,   104 
Cubic  centimeter,  4 
Culture  media,   166 

agar-agar,   169 

blood  serum,  95,   170,  173 
Gibson's  modification  of, 

i74 
bouillon,    169 

sugar  free,  170,  174 

sugar,    1 74 
glucose   agar,    169 
glucose  bouillon,   169 
glycerin  agar,   169 
Clegg  and  Musgrave,  for 

ameba,   175 
differential,    175,    178 
egg,   171 
Endo's,    172 
gelatin,    170 


Culture  media,  Hesse's,  176 
Hiss,   175 

Jackson's  lactose  bile,   173 
litmus  milk,   171 
potato   slant,    177 

glycerin   potato,    177 
reaction,  166,  167 
special  for  diphtheria,  171 
for  gonococci,   173 
for   rapid   culture    of   B.   tu- 
berculosis,   178 
titration,    167 
Culture  taking,   144 
Curds,    68 

Curschmann'-s  spirals,   84 
Cylindroids,  45 
Cystin,    38,    40 
Cytodiagnosis,   182 
Cytolysis,    129 

Decalcification,   195 
Decimal  table,  5 
Delafield's  method,   193,  204 
Diacetic  acid,  35 
Diastase,   61 
Diazo   reaction,   36 
Diets,  diabetic,  54 

fat   free,  68 

nephritic,  52,  53 

salt   free,  52 
Dimethyl-amido-azo-benzene 

(Topfer's  reagent),  71 
Dittrich's  plugs,  84 
Duboscq,  directions  for  using, 
137 

Echinococcus,  46 

Eels  in  urine,  46 

Elements,  9 

Endospores,   144 

Endothelial  cells,   181,   182,   183 

Eosinophiles,    105,    106 

Epithelium,   in  sputum,  85 

in  urinary  sediment,  41 
Equivalents,  table  of,  7 
Erythroblasts,  93 
Erythrocytes,     counting    of,    101 

crenated,    104 

number  per  cubic  mm.,  104 

Wright's  stain,  104,   105 
Esbach's  albuminometer,  27 

reagent,  preparation  of,   198 


Index 


209 


Ewald's  test   meal,    77 
Exudate,  180 


Fahrenheit  scale,   13,   14 

False    casts,    45 

Fasting  contents,   macroscopic 

examination   of,    77 
microscopic  examination   of, 

77 

qualitative   examination  of,  77 
quantitative     examination     of, 

78 
Fat,  in  feces,  58 

in  milk,  191 
Fatty  acids,  58 
Fatty  casts,  in  urinary  sediment, 

44 

Fecal  bacteria,   62 
Feces,   bile,    60 

blood,  59,  67 

color,   56,  67 

consistency,  57 

daily  excretion,  56 

detection  of  bilirubin,  60 

diastase,  61 

eggs,  examination  for,  64 

Fat  and  fatty  acid,  58 

food,   58 

hydrobilirubin,   60 

micro-chemical   examination, 

57 

micro-organisms,  62 

mucus,    58,    67 

odor,   57 

ova,   64,    65 

parasites,  62,   63 

pus,   6 1 

reaction,   57 

scybalous  masses,   57 

soaps,    58 

trypsin,   61 

tubercle   bacilli,   62 
Fehling's  solution,   198 
Fermentation  test,  29,  32 
Ferric  chloride  test,  73 
Filaria  Bancrofti,  121 

sanguinis  hominis,   121 
Folin's  method,  blood  urea,   134 

determination    of    non-protein 
nitrogen,   132 

sugar  in  blood,   137 


Foreign     substances,     in    micro- 
scopical   examination,    18 
in  urinary  sediment,   21 
Formaldehyde,  excretion  of,  46 

fixation  in  tissue,   192 
Free    hydrochloric    acid,    71,    72, 

81 

Free  nucleus,  105 
Fuchsin,   reagent,  preparation  of, 
203 


Gabbet's  stain,  203 
Gastric  contents,  70,  77 

blood,  73,   74 

carcinoma,  80 

character,    70 

color,   70 

combined  acidity,  81 

food,   73 

hydrochloric  acid,   71,  72,  75 

lactic  acid,  72,  73 

mucin,   72 

organic  acid,  72,  82 

pepsin,  73 

poisons,   75-77 

rennin,   73 

total  acidity,  79,  80 
Gelatin  media,    170 
Gerhardt's  test  for  acetone,   34 
Giemsa's  stain,  204 
Glass,  composition,  I 

droppers,  2 

flasks,    1 1 

pipettes,  2,  ii 

rods,  2 

slides,   i 

stoppers,  to  remove,  6 

tubes,   i 
Glassware,  care,  i 

cleaning,  2 

Glossary  of  terms,   161 
Glucose,  agar-agar,  169 

bouillon,   169 

Benedict's  test  for,  qualitative, 
29 

quantitative,   31 

in  urine,   28-33 
Glycerine    agar-agar,    169 
Gmelin's  test,  60 
Gold   solution,    preparation  of, 
1 86 


2IO 


Index 


Goodman   and    Steam's   method, 

28 

Gonococci,   144,   150 
Gower's  solution,   197 
Gram's  stain,   153,  183,  202 

preparation  of,    202 
Gram's   codine   solution,  202 
Granulomatoses,    109 

Smith's  simplification  of,   153 
Guaiac  test,  in  feces,  59 

in  gastric  contents,   73 
Gunzburg's    reagent,    preparation 
of,   202 

test,   72 

Halliburton's  table,  22 

Hanging    drop,    preparation,    143 
slide,    117 

Haymen's     solution,    preparation 
of,   197 

Heat  test,   for  albumin,   26 

Heller's    test,    for   albumin,  26 

Hematoblasts,  94 

Hemacytometers,    97 

Hemoglobin,  90,  96,  97 

Hemolysis,   91,    112,   129 

Hematoidin  crystals,  40 

Hodgkins   Disease,   108 

Howell-Jolly  bodies,  93 

Hyaline  casts,  43 

Hydrobilirubin,    detection    of,    in 
feces,    60 

Hydrocele,    183 

Hydrochloric  acid,    75,   81 

estimation    of,   in   gastric    con- 
tents,  71,   78  8 1 

Hydrogen  peroxide,  detection  of, 
in  milk,    190 

Hydruria,  20 

Hyper-acidity,   71 

Hyper-chlorhydria,    71 

Hypo-acidity,  71 

Hypo-chlorhydria,  71 

Immune  bodies,    129  , 

Inactivation,   129 

Indican,  test  for,  35 

Indole,   57 

Indoxyl,   35 

Infants'  stools,   66 

Infusion,   166 


Iodine  test,  36 
Jackson's  bile  media,  173 

Laboratory  equipment,   i 

rules,   14 
Lactic   acid,   in  gastric  contents, 

72,  73 
Lactose,  29 
Laked  blood,  91 
Lange's  test,  184 
Leache's  test,    190 
Lead,  test  for,  27 
Lesions  caused  by  bacteria,  151 
Leucin  crystals,  in  urinary  sedi- 
ment, 38 
Leucocytes,    94,    105 

in  body  fluid,  183 

counting,    100,   184 

function,    94 

number  per   cubic  mm.,    101 

in  sputum,  85 

in  urine,  42,  43 

Wright's  stain,    104,   105 
Leucocytosis,   94,    109 
Leucopenia,  94 
Leukemia,   lymphatic,    108 

myeloid,   108 
Lipo-vaccines,    147 
Litmus  milk,   171 

paper,   23 

reaction,   23,    166 

serum,   170 
Loffler's  methylene  blue,  203 

stain,    for  diphtheria,    154 

preparation  of,  203 
Lugol's   solution,   preparation   of, 

202 

Lymphocytes,    105 
Lymphoid   pseudoleukemia,    109 
Lymphosarcoma,   109 

Macrocyte,   104 

Magnesium  mixture,  26,  199 

preparation  of,   199 
Malarial  parasites,   118-121 
Mast  cells,  105,  106 
Mayer's  albumin  solution,  194 
Measures,  4 
Mechanical  stage,   103 
Meconium,    66 
Media,    166 


Index 


211 


Media,  agar-agar,  169 

glucose,    169 

glycerin,   169 
bile,    173 
blood  serum,   170 

substitute  for,   173 
bouillon,   169 

glucose  in,   169 

sugar  in,   174 

sugar   free,   170,   174 
differential,    175,    178 
egg,   171 
Endo's,    172 
gelatin,    170 

Gibson's    modification    of    sub- 
stitute   for    blood    serum, 

174 

glycerin   potato,    177 

Hesse's,    176 

Hiss,    175 

lactose,    173 

litmus  milk,   171 

Musgrave   and   Clegg,    175 

potato   slants,    177 

reaction,    166,   167 

special  for  ameba,  175 
diphtheria,    171 
gonococcus,    173 
rapid  culture  of  B.  tubercu- 
losis,   178 

titration,  167 
Megacaryocyte,   107 
Megaloblast,    105 
Melangers,    98 
Melanin  in  urine,  36 
Methylene    blue,   preparation    of, 
saturated  solution,  6 

Loffler's  stain,   203 
Micrococci,    142 
Micrococcus  urese,  45 
Micron,  142 
Micro-organisms,   in  feces,  60-62 

in   urine,   45 
Microscope,    16 

care,    18,    19 

construction,   16,   17 

focusing,    1 7 

illumination,  17 
Microtome,    192,    196 

knife,    192 
Milk,    187 

bacteria,    188 


Milk,  counting  of,    191 

borates,    boric    acid,    detection 
of,  190 

butyric   acid,    184 

caseinogen,    188 

cream,  187 

definition  of  terms,    187 

difference  between  human  and 
cow's,  187 

fat,   191 

fermentation,   188 

formaldehyde,    detection    of, 
190 

hydrogen    peroxide,     detection 
of,    190 

Pasteurization,   189 

preservatives,    189 

reaction,    187 

salicylates,    salicylic    acid,    de- 
tection of,   190 

specific  gravity,   187 

sterilization,    188,    189 

typhoid,    191 

whey,   1 88 
Mitochondria,  102 
Molds   in  urine,   46 
Mononuclears,    105,    106 
Mounting,    196 
Mucin,    72 
Mucous   shreds,   45 
Murexid   test,   37 
Myeloblasts,   107 
Myelocytes,  basophilic,   105,   106 

eosinophilic,    105,    106 

neutrophilic,    105,    106 
Myelogenous  leukemia,    108 
Myeloma,    multiple,    109 

Nakanishi's   method,    143 
Neisser's    stain,    preparation    of, 

203 

Newton's  rings,  99 
Nicolle's  method,    195 
Nitric  acid,   75 

test,   26 

Nonne's  globulin  test,   182 
Normal    urine,    20 
Normoblast,   105 
Nucleated  red   cell,    105 
Noguchi's     serum     diagnosis     of 

syphilis,    122 
Nylander's  reagent,   199 


212 


Index 


Occult   blood,   test   for,   in  feces, 

59 

gastric  contents,   74 
Oleum  origani  cretici,   193 
Oligochromenia,  97 
Oligocythemia,  92 
Oliguria,   20 
Organized     sediment,     in     urine, 

4i 

Oxalic   acid,   75 
Oxydase    reaction,    in    leukemia, 

109 
Oxyuris  vermicularis,   62,  65 

Pancreatic  insufficiency,  62 
Parasites,  46,   64,  121 

malarial,    118-121 
Para-typhoid,  "type  A,"   116 

"type    i>,"    116 

Pasteur's    method    of    cultivating 
anaerobes,  145 

preserving  milk,   189 
Pentose,  29 
Pepsin,    73 

Pericardial  fluid,   181 
Peritoneal  fluid,   182 
Petri  plates,  147 
Phenolphthalein  test,    60 
Phe-nolsulphonephthalein  func- 
tional   test,    50 
Phenylhydrazin   test,   29,   32 
Phosphates,  39,  41,  47 
Phosphotungstic  solution,  199 
Platelets,    114,    115 
Platinum  loop,    15 
Pleural   fluid,    181 
Pneumococcus,   86,   87,   149 
Poikilocytes,    105 
Poisons,  tests  for,  alkaloids,  76 

arsenic,  74 

belladonna,  76 

carbolic  acid,  74 

chloral,    75 

corrosive  sublimate,  75 

hydrochloric  acid,   75 

nitric  acid,  75 

oxalic  acid,  75 

opium,  77 

phosphorus,  76 

prussic   acid,   76 

strychnine,   77 


Poisons,  sulphuric  acid,  76 

Polarimetric  test,  29 

method     of     determination     of 
glucose  in  urine,  29 

Polychromatophilia,   92,    105 

Polychrome    methylene    blue, 
preparation  of,  205 

Polycythemia,   92 

Polymorphonuclear  neutro- 
phiks,    105,    1 06 

Potassium      sulphocyanide      solu- 
tion,   199 

Preservatives,   in  milk,   189 

Pseudoleukemias,    108,    109 

Purulent  pleurisy,   181 

Pus,  in  feces,  61,  67 
in  urine,  43 

Qualitative   analysis,   of   feces, 

56-68 

of  gastric  contents,  70-88 
of  urine,  25-30,  45,  48 
Quantitative    analysis, 

of  gastric  contents,   78,   79-82 
of  urine,  27,  31-37,  47-5* 
albumin,  27,  28 
ammonia,  49 
chlorides,  48 
phosphates,   47 
sugar,  30-32 

Raw  and  heated  milk  test,  189 
Reaction,  in  blood,  91 
in  feces,  57 
milk,    187 
urine,    23 
Reagents,   5 

for  blood,  197,  198 

gastric   contents,   201,   202 
liquid,  5 
solid,  5 

stains,  202,  203,  204 
urine,    198-201 

Red  blood  corpuscles,  appear- 
ance, 42,  43 
counting    of,    101 
number  per  cubic  mm.,  104 
Register    for    recording    corpus- 
cles,  100 

Reinsch's   test  for   arsenic,   74 
Renal  functional  test,  50 
Rennin  test,   73 


Index 


213 


Resorcin  test,  72,  201 
Ringer-Locke    solution,    prepara- 
tion of,   102 

reagent,  preparation  of,  201 
Rowntree  and  Gerahty's  renal 
function   test,   50 


Sahli's  hemoglobinometer,   96 
Salkowski's   test,   35 
Sarcinae,  142 
Saturated  solution,  8 
Scale,   Centigrade,  14 

Fahrenheit,    14 
Sediments,    urinary,    38,    39,   40, 

4i, 
Serum,  albumin,  25 

globulin,    25 
Silver  solution,  200 
Skatole,   57 
Slides,    i 

hanging  drop,  117 
Smears,  blood,  103 

body  fluids,   183,   184 

feces,   60 

sputum,  85,   86 
Smegma  bacilli,   156 
Soaps,    58 

Sodium    acetate    solution,    199 
Solids,    estimation    of,    in    urine, 

24 
Solutions,  approximate,  7 

concentrated,   8 

normal,  8 

standard,   8,    199,    200 

vaccine,   146 
Spermatocele,    183 
Spermatozoa,   45 
Sphygmomanometer,    "Tycos," 

130 

Spirilla,  141 

Spirochetae,  stain  for,   158 
Sprue,   57 
Sputum,  bacteria  in,  85 

color,   84 

consistency,   84 

elastic   fibers,   85 

epithelium,   85 

erythrocytes,  85 

macroscopical,  84 

microscopic  examination,  85 


Sputum,  odor,  85 

quantity,    84 

reaction,  85 

Squamous  cells,  in  urine,  42 
Stains,   alum-hematoxylin,   204 

Delafield's,    204 

Gabbet's    156 

Giemsa's,   158 

Gram's,   153 

India  ink,  159 

Loffler's,    154,   157 

Mallory's,  159 

Moeller's,   157 

Neisser's,   156 

Polychrome   methylene   blue, 
205 

Smith's  modification    of 
Gram's  85 

Smith's    simplification    of 
Gram's,   153 

Van  Gieson's,    195 

Welch's,  157 

Ziehl-Neelsen's,  86,   156 
Staining  methods,   152 
Staphylococcus  albus,    149 

pyogenes  aur~us,   149 

pyogenes  citreus,   149 
Stasis,  71 
Steatorrhea,  57 
Stippled  cells,  93 
Streptococcus   hemolyticus,   148 

pyogenes,    149 
Sugar,   28 

in  blood,  137 
Swabs,    144 
Symbols,   9 
Synovial  fluid,  181 
Syphilis,   122,  152,   158 

Table   of  equivalents,   7 
Talquist's   scale,   97 
Tissue,   192 

collodion  method,  174 
connective,   56 
elastic,  85 

fixatives,  alcohol,   192 
corrosive  sublimate,   192 
formaldehyde,    192 
Zenker's  fluid,   192 
freezing  method  for  rush 
diagnosis,   192 


214 


Index 


Tissue  mounting,   196 

preparation   of   fresh,    194 

staining,  195 
Titration,   in  adjustment  of,    167 

reaction  in  media,   167 

normal  solutions,  10 

standard  solutions,  12,  199,  200 
Toisson's  reagent,  197 
Topfer's  reagent,  71 
Total   acidity,   in  gastric   con- 
tents, 79,  80 
Transfusion,  matching  bloods 

for,    in 

Transudate,   iC  > 
Triple    phosphates,    crystalline 

form,  38 

Trypanosomes,    100 
Trypsin,  61 

Tryptophane  test,  80,  82 
Tubercle  bacilli,  86,  150 

in    feces,    62 

staining,  87 

in  urine,   45 

Turbidity,  in  urine,  20,  26 
Turk's  irritation  form,   109 
"Tycos"  apparatus,  130 
Typhoid,  agglutination,   115 

differential  media,   118 

Hesse's  media  for,  176 

para-typhoid,    types    "A"     and 
"B,"   116 

Widal   reaction,   115,   116 
Tyrosin,   38 

Uffelmann's  test,   72,   202 
Uranium  acetate   solution,    199 
Urates,  ammonium,   38 

sodium,    38 
Urea,  nitrate,  37 
Uric  acid  crystals,  38-41 
Urinary  sediments,  38 
Urine,   acetone,   21,  34 

acidity,  23,  48 

albumin,    25 

alkaline  tide,  23 

Alkapton,   23 

ammonia,  20,  49 

aromatic  acids,  20 

bacteria,  20,  45 

bile,    36 

blood,   34 

collection  for  analysis,  21 

color,  21,  22 


Urine,  consistency,  20 

creatinine,  37 

diacetic  acid,  35 

globulin,   25,   26 

indican,    35 

Long's    coefficient    for    estima- 
tion of  solids,  24 

melanin,    36 

odor,  20 

pentose,    29 

phosphates,  39,  41,  47 

pigments,   37 

reaction,    23 

specific  gravity,  24 

sugar,  28,  29,  30,  31,  32,  33 

urates,   38,  40 

urea,    37 

uric  acid,   37 

urobilin,    36 

urochrome,   37 

urophaein,  37 
Urochromogen  test,  36 

Vaccines,  bacterial,   145 

dosage,   129 

preparation  of,  145 

special  diluting  fluid,    146 

standardization,  146 
Van  Gieson's  stain,   195 
Vital  staining,  of  blood,   102 
Volhard-Harvey  method,  48 
Vomitus,  70 

Wassermann   reaction,    121 

test,    122 

Water,  at  meals,  influence  of,  62 
Waxy  casts,   45 
Weights,    2 
Weyl's  test,    37 
Widal  Reaction,   115,   116 
Wright's    method    of    cultivating 

anaerobes,  145 
Wright's  stain,   104,   105 

preparation  of,  198 

Xylol,    for    cleaning    slides,    2 
clearing  agent,   193 
balsam,  196 

Yeast  fungi,  46 

Zenker's   fluid,   preparation   of, 
195 

Ziehl-Neelsen    method    of    stain- 
ing,   87 


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r-i?    o  jr-}5 

MAR  121935 


21  1937 
SEP.1°19«2 


EJ 


LD  21-50?n-8,-32 


